- Support - Troubleshooting Guide

1. Incomplete digestion or no digestion

1.1. Inactive enzyme

If the enzyme does not cut the control DNA:

Check the expiration date.
Verify that the enzyme has been stored at -20°C.
Check the temperature of your freezer. Do not allow the temperature go below -20°C as the enzyme may freeze and multiple freeze thaw cycles (more than 3 cycles) may result in reduced enzyme activity.

1.2. Suboptimal digestion protocol

Follow digestion protocol specified for the restriction enzyme and type of substrate DNA.

Use the recommended reaction buffer supplied with the restriction enzyme. For double digestions with conventional restriction enzymes, follow the recommendations of the DoubleDigest™ engine.
Use additives where required.
Perform the reaction at the optimal temperature specified for the restriction enzyme. For double digestions with mesophilic and thermophilic conventional restriction enzymes, first digest with the mesophilc enzyme (1 h), then increase the temperature and incubate for an additional hour.
Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the increase in salt concentration may reduce enzyme activity. For thermophilic enzymes use a heat block with a hot bonnet, e.g. a PCR cycler.

1.3. Improper enzyme dilution

Dilute restriction enzymes with Dilution Buffer for Restriction Enzymes. Restriction enzymes diluted with this buffer are stable for at least 3-4 weeks at -20°C.
Never dilute enzymes in water or 10X reaction buffer.
Never dilute enzymes in 1X reaction buffer in the absence of DNA.

1.4. Improper reaction assembly

The restriction enzyme should always be the last component added to the reaction mixture.
The restriction enzyme may be inactivated if added directly to a 10X reaction buffer.

1.5. Excess glycerol in the reaction mixture

The glycerol concentration in the reaction mixture should not exceed 5%. Thus, the volume of the restriction enzyme added to the mixture should not exceed 1/10 of the total reaction volume.
Enzymes sensitive to high glycerol concentration include: Alw21I, BpiI, Bsp68I, BspTI, Eco32I, Eco91I, EcoRI, Hin6I, HinfI, Mph1103I, Mva1269I and NcoI.

1.6. Suboptimal DNA concentration

The optimal range of DNA concentration in the reaction mixture is 0.02-0.1 µg/µl.

1.7. Unsuitable DNA template or contaminated DNA solution

If the enzyme is active in the control digest, assay the substrate DNA solution for inhibitory contaminants in a mixing experiment with control template, e.g. Lambda DNA (dam-, dcm-). Perform a control digest with two templates: control template and sample template in one reaction mixture. Do not exceed the optimal DNA concentration in the reaction mixture (0.02-0.1 µg/µl).

The sample template is contaminated if neither the control DNA template nor sample template is digested. (see 1.8).
The sample template is not contaminated if the control DNA template is digested but the sample template is not. Poor digestion of the experimental template is caused by errors in the DNA sequence (see 1.9), methylation effects (see 1.10) or structure of the DNA substrate (see 1.11).

Note

Always ensure that the control DNA contains a recognition site for the enzyme present in the reaction. For example, there is no NotI recognition site in lambda DNA.

1.8. Contaminants in the DNA solution

Template DNA may contain residual SDS, EDTA, proteins, salts or nucleases. Repurify the template using a spin column purification kit or by phenol/chloroform extraction and ethanol precipitation. DNA A₂₆₀/A₂₈₀ ratio should be 1.8-2.0. To remove EDTA and salts, wash the pellet with 70% cold ethanol.
For reliable and reproducible plasmid miniprep purity, use the GeneJET™ Plasmid Miniprep Kit.
For digestion of unpurified PCR products, dilute DNA at least 3-fold in the recommended 1X restriction enzyme buffer.
If the template DNA has been purified using silica or resin suspensions, remove all remaining particles by centrifugation for 10 min at 10,000 rpm and ensure that no resin is carried over while transferring the DNA solution into a new tube.

1.9. The substrate DNA does not contain a recognition sequence for the restriction enzyme

Re-check the DNA sequence and cloning strategy.
Determine if the restriction enzyme selected requires more than one site per target DNA for 100% activity (see also 1.11.3).
Check literature for known site preferences for the restriction enzyme (see also 1.11.4).
If the recognition sequence had been introduced by PCR primers, verify that the primer sequence contains the recognition site.

1.10. Methylation effects

Restriction enzyme is inhibited by methylation of the recognition site.

Identify which type of DNA methylation can occur on the recognition site and determine if the methylation impairs or blocks DNA digestion with the enzyme. See Digestion of Methylated DNA.
If methylation impairs or blocks DNA cleavage:
- propagate your plasmid in an E.coli dam-, dcm- strain (the E.coli GM2163 dam-, dcm- strain; #M0099, is available upon request with the purchase of any Fermentas product),
- use the REsearch™ engine or check the Fermentas catalog for the availability of a restriction enzyme isoschizomer not sensitive to DNA methylation.

A restriction enzyme which requires a methylated recognition sequence (DpnI) was used to digest unmethylated DNA.
In the case of DpnI, the neoschizomers Bsp143I or MboI can be used to digest non-methylated DpnI recognition sites. Alternatively, propagate your plasmid in E.coli dam+ strains (most conventional laboratory strains are dam+).

Note.

When PCR is carried out with standard dNTPs and non-methylated primers the resulting DNA product is NOT methylated.

1.11. Structure of substrate DNA

1.11.1. Supercoiled plasmid DNA.
Use FastDigest® enzymes which are qualified for supercoiled DNA and provide specific recommendations for each enzyme.
For some conventional restriction enzymes, additional units are required to digest supercoiled plasmids completely (e.g. 5-10 u (1 µl) of restriction enzyme per 1 µg of DNA), refer to the Certificate of Analysis.

1.11.2. Proximity of the recognition sequence to the DNA ends.
Some restriction enzymes cleave DNA poorly, if the recognition site is too close to the end of the DNA molecule.

For FastDigest® enzymes refer to the product description to determine the effectiveness of restriction enzymes at the ends of DNA.
For conventional restriction enzymes refer to Table "Cleavage Efficiency Close to the Termini of PCR Fragments" and Table "Cleavage of Restriction Targets Located in Close Vicinity within pUC Multiple Cloning Site"
Consider direct cloning of your PCR product into a cloning vector, e.g. CloneJET™ PCR Cloning Kit or InsTAclone™ PCR Cloning Kit (available in certain countries only).

1.11.3. Restriction Enzyme requires at least two sites per DNA molecule to obtain optimal activity.
Some restriction enzymes such as AarI, BveI, Cfr42I, Eam1104, Eco57I, EcoRII, LweI, SfiI require at least two target sites per DNA molecule for efficient cleavage. If there is only one recognition site per DNA molecule, add a DNA oligonucleotide containing the recognition site.

1.11.4. Site Preferences by Restriction Enzymes.
The DNA sequence surrounding the recognition site may influence the efficiency of digestion. Some DNA sites are cleaved slowly or not cleaved at all due to the surrounding sequence. Use additional units (5-10 u) of the restriction enzyme per 1 µg of DNA or determine if an isoschizomer has superior cleavage efficiency (see REsearch™ engine).

1.12. Water contains impurities

Compare you results using commercially available nuclease free, molecular biology grade water, e.g. Water, nuclease-free. Check the quality of the water used in you lab.

Check the pH and conductivity of water. The pH of high quality water should be 5.5-6.0.
Centrifuge (10 min, 10,000 rpm) 1 ml of water and check if there is a visible pellet.
Determine if the water contains nucleases or bacterial contamination (see 3.2 for control reactions).

2. Unexpected cleavage pattern

2.1. Star activity (relaxed specificity) of restriction enzyme

Reduce the units of restriction enzyme (not more than 10 u of restriction enzyme or 1 µl of FastDigest® restriction enzyme per 1 µg DNA).

Use the recommended reaction buffer.

Ensure that the glycerol concentration in the reaction mixture does not exceed 5%.

Reduce the incubation time.

Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the resulting increase in glycerol concentration may cause star activity.

2.2. Contamination with another restriction enzyme

The restriction enzyme or buffer may be contaminated with another restriction enzyme due to improper handling. Use a new tube of enzyme and/or buffer.

2.3. Contamination with another substrate DNA

The sample DNA contains a mixture of two or more different DNAs. Prepare new sample of DNA.

For plasmid DNA preparation pick one isolated colony of recombinant E.coli and purify with GeneJET™ Plasmid Miniprep Kit.
For PCR products: check the product purity on an agarose gel. If necessary, purify the PCR product prior to digestion with the DNA Gel Extraction Kit.

2.4. Incomplete DNA digestion

Different DNA structures like nicked, supercoiled, dimeric molecules will always show different mobility on gels compared to same size DNA size standards, as an example, see the picture below for migration of plasmid DNA forms:

Figure. Migration of plasmid DNA forms

GeneRuler™ 1 kb DNA Ladder.
Undigested plasmid pUC19 2,7 kb DNA, forms:
- upper band (~5 kb) – dimeric plasmid,
- below, less visible (~4 kb) – nicked plasmid,
- lowest band (~1.9 kb) – supercoiled plasmid.
Linearized plasmid pUC19 (2,7 kb) – migrates according to its size.

2.5. Unexpected recognition sites in template DNA

Newly generated target sites in constructed DNA may be overlooked. Recheck your DNA sequence and cloning strategy.

3. Diffused DNA bands

3.1. Gel shift

Enzyme that remains bound to the substrate DNA will affect the electrophoretic mobility of the digestion products. Restriction enzymes AarI, AloI, BdaI, BseXI, BveI, CseI, Eco57I, Eco57MI, EcoRII, FaqI, GsuI, LweI, MboII, FastDigest® MboII, MnlI, FastDigest® MnlI, SchI, TsoI, TstI are particularly prone to remaining bound to the substrate DNA. This will result in a band or smear above the expected band (see picture below). Use 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of 1X SDS prior to electrophoresis.

M – GeneRuler™ DNA Ladder Mix
1 – 0.5 µg lambda DNA prepared for loading with 6X DNA Loading Dye
2 – 0.5 µg lambda DNA prepared for loading with 6X DNA Loading Dye & SDS Solution
3 – 0.5 µg lambda DNA digested with TsoI, probe prepared for loading with 6X DNA Loading Dye
4 – 0.5 µg lambda DNA digested with TsoI, probe prepared for loading with 6X DNA Loading Dye & SDS Solution

3.2. Contaminated reagents

Any restriction digestion reaction components may become contaminated with nucleases due to improper handling or storage. Nuclease contamination causes DNA degradation, which appears as diffused DNA bands on a gel.
Perform four control reactions:
I – without restriction enzyme,
II – with a new vial of buffer,
III – without restriction enzyme, with a new vial of buffer,
IV – with commercially available water e.g. Water, nuclease-free.

Contaminated sample DNA (diffused bands in all controls). Repurify the DNA sample by spin column or phenol/chloroform extraction and ethanol precipitation.

Contaminated enzyme (diffused bands in controls 2 and 4). The enzyme may become contaminated due to improper handling. Use a new vial of enzyme.

Contaminated buffer (diffused bands in controls 1 and 4). Bacterial contamination of the reaction buffer will cause DNA degradation. Use a new vial of buffer. Store all buffers at -20°C.

Contamination of both enzyme & buffer (diffused bands in controls 1, 2 and 4). Follow the recommendations given above.

Contaminated water (diffused bands in controls 1, 2 and 3). Bacterial or DNase contamination in improperly handled water will cause DNA degradation. Use commercially available nuclease free molecular biology grade water.

- Troubleshooting Guide for DNA Digestion in PDF

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1. Low yield or no PCR/RT-PCR product

1.1. Poor template integrity.

DNA templates. Evaluate template integrity by agarose gel electrophoresis. Use DNA isolation methods that minimize shearing and nicking of DNA. Resuspend isolated DNA in TE buffer, pH 8.0, or in Water, nuclease-free.
RNA templates. RNA purity and integrity is essential for synthesis of full-length cDNA, which results in high quality RT-PCR products. Always assess the integrity of RNA prior to cDNA synthesis. For example. if sharp bands of both the human 18S rRNA (runs at approx. 1.9 kb) and the 28S rRNA (runs at approx. 5 kb) are formed during denaturing agarose gel electrophoresis of total human RNA, the mRNA in the sample is intact. Follow general recommendations to avoid RNase contamination.

1.2. Low template purity

DNA templates. Generally most commercially available DNA purification methods yield template suitable for PCR, e.g. Genomic DNA Purification Kit or GeneJET™ Plasmid Miniprep Kit. However, trace amounts of certain agents used in home-made DNA purification protocols, such as phenol, EDTA, and Proteinase K may inhibit thermostable DNA polymerases. In addition, high ionic concentrations (e.g. K⁺, Mg²⁺, etc.) may lead to suboptimal reaction conditions for the DNA polymerase. In cases such as this, the template should be re-purified using using a spin-column or re-precipitated and washed with 70% ethanol.
RNA templates. Trace amounts of agents used in RNA purification protocols may remain in solution and inhibit reverse transcriptases, e.g. SDS, EDTA, guanidine salts, phosphate, pyrophosphate, polyamines, spermidine. Precipitate the RNA with ethanol and wash the pellet with 75% ethanol.

1.3. Low quantity of template

DNA templates. Increase the amount of template or use hot start enzymes or master mixes, e.g. TrueStart™ Hot Start Taq DNA Polymerase, Maxima® Hot Start Taq DNA Polymerase or PyroStart™ Fast PCR Master Mix (2X). Alternativley, use enzymes or master mixes with higher sensitivity than that of Taq DNA Polymerase, e.g. DreamTaq™ DNA Polymerase or High Fidelity PCR Enzyme Mix.
RNA templates. Increase the amount of template to the recommended level. After DNaseI treatment, terminate the reaction by heat inactivation in the presence of EDTA. Heat inactivation in the presence of divalent cations degrades RNA.

1.4. Faulty primer design

DNA templates. Use REviewer™ primer design software. Make sure primers are not self-complementary and avoid between primer complementary sequences. Extension of primer duplexes will consume reaction components and result in lower yields of the target PCR product. Verify that the primers are complementary to the correct strands of template DNA.
RNA templates. Use the correct primer for the type of RNA template used for reverse transcription. Do not use the oligo(dT)₁₈ primer for bacterial RNA or RNA without poly(A) tail. In such cases the random hexamer primer is recommended. If using sequence-specific primer, ensure it is complementary to 3'-end of the template RNA.

1.5. Suboptimal thermal cycling conditions

Follow the general PCR cycling recommendations for the specific DNA polymerase you use.

1.6. Annealing

The annealing temperature should be 5°C lower than the primer-template melting temperature (Tm). The annealing temperature may be optimized stepwise in 1-2°C increments. If available, use a gradient cycler to optimize the annealing temperature of a specific primer pair (±10°C).
The annealing temperature also has to be adjusted when additives that change the melting temperature of the primer-template duplex are used, e.g. glycerol, DMSO, formamide, betaine, TMANO (trimetylamine-N-oxide).

1.7. Extension

The recommended reaction temperature is 72°C for Taq and Pfu DNA polymerases. As a general rule, the extension step with Taq DNA Polymerase takes 1min/kb. As Pfu DNA Polymerase has a lower extension rate, allow 2 min/kb for extension step with this enzyme. For fast cycling PCR, e.g. using Fermentas PyroStart™ Fast PCR Master Mix (2X), the extension time can be shortened to 25 s/kb and lower.
For amplification of longer templates (>3 kb) the extension temperature can be reduced to 68°C. Long PCR Enzyme Mix and High Fidelity PCR Enzyme Mix are ideal for such applications. In addition to the standard extension time (1 min/kb) an auto-extension per cycle is recommended for later cycles when amplifying longer templates.

1.8. Number of Cycles

The number of cycles varies depending on the amount of template DNA in the PCR mixture and the expected yield of the PCR product. Generally 25-35 cycles are sufficient to produce an adequate yield of PCR product. If less than 10 copies of the template DNA are present in the reaction, extend the number of cycles to 40.

1.9. Insufficient amount of DNA polymerase

For a 50 µl PCR mixture, we recommend adding 1-1.5 u of Taq DNA Polymerase or 1.25-2.5 u of Pfu DNA Polymerase. However, it may be necessary to increase the amount of DNA Polymerase, if the PCR mixture contains inhibitors, due to contamination of the template DNA.

1.10. Insufficient amount of primer

Generally the PCR reaction is successful with wide range of PCR primer concentrations (0.1-1 µM) and the optimal conditions will vary depending on specific primer/template pair. A primer comcentration of 0.4 µM is a good starting point for optimization. For long PCR and PCR with degenerate primers a minimum of 0.5 µM is recommended. Also, assay primer degradation on a denaturing polyacrylamide gel.
PCR primers may degrade due to the 3'=>5' exonuclease activity of Pfu DNA Polymerase or PCR Enzyme Mixes. Therefore, PCR mixtures should be kept on ice during the reaction set-up and the polymerase or mix should be the last component added to the reaction mixture. Alternatively, phosphorothioate primers can be used to avoid primer degradation by Pfu DNA Polymerase.

1.11. Insufficient Mg²⁺ concentration

If the Mg²⁺ concentration is too low, the yield of PCR product may be reduced. Due to the binding of Mg²⁺ to dNTPs, primers and DNA template, Mg²⁺ concentration often needs to be optimized for maximal PCR yields. The recommended concentration range for optimizations is 1-4 mM.
For standard PCR with 0.2 mM dNTP and Fermentas Taq DNA Polymerase, a good starting MgCl₂ concentration is 1.5 mM (for Taq buffer with KCl) and 2.0 mM (for Taq buffer with (NH₄)₂SO₄). For Pfu DNA Polymerase we recommend a starting concentration of 2 mM of MgSO₄ and for DreamTaq™ DNA Polymerase – 2 mM of MgCl₂.
If template DNA contains EDTA or other metal chelators, the Mg²⁺ ion concentration in the PCR mixture should be increased accordingly (1 molecule of EDTA binds 1 molecule of Mg²⁺).
In certain PCR applications higher dNTP concentrations are required. dNTPs also complex Mg²⁺, therefore the Mg²⁺ concentration has to be increased accordingly.

1.12. dUTP or modified nucleotides in reaction mix

Proofreading polymerases or enzyme mixes containing such proofreading polymerases may incorporate dUTP with much less efficiency compared to standard dNTPs. If possible, use non-proofreading polymerases like Taq DNA Polymerase to incorporate dUTP into the PCR product. Alternatively, use a higher ratio of dUTP:dNTP to achieve the desired yield of labeled PCR product.

1.13. GC-rich template

DNA templates. If the template has high GC content, and/or forms a complex secondary structure, we recommend using our Long PCR Enzyme Mix. Alternatively DNA denaturation can be enhanced by the addition of either 10-15% glycerol, 10% DMSO or 5% formamide. When any of these additives are used, the annealing temperature has to be lowered. Since DMSO and formamide inhibit polymerases by approximately 50%, the amount of the enzyme in the PCR mix also has to be increased.
RNA templates. If the RNA template is GC rich or known to contain secondary structures, use reverse transcriptases with high thermostability, e.g. AMV Reverse Transcriptase or RevertAid™ H Minus Reverse Transcriptase, and increase the temperature of the reverse transcription reaction.

1.14. Long template

Use appropriate long PCR enzymes for templates longer than 3 kb, e.g. High Fidelity PCR Enzyme Mix or Long PCR Enzyme Mix. The extension temperature can be reduced to 68°C. In addition to the standard extension time (1 min/kb) an auto-extension per cycle is recommended for later cycles when amplifying long templates.

2. Non-specific PCR products

2.1. Faulty primer design

Use REviewer™ primer design software.
Verify that the primers are complementary to correct strands of template DNA.
Verify that the primers are specific to the template region selected for amplification and have no complementarity with other regions in the template DNA. Otherwise, primers will anneal nonspecifically and generate unexpected PCR products.
Ensure primers are not self-complementary contain between primer complementary sequences. Otherwise extension of primer duplexes will generate unexpected products.
Avoid direct repeats in the primers to limit the appearance of large PCR products compared to the target amplicon.

2.2. Reaction set up at room temperature

When a PCR reaction is set up at room temperature, Taq DNA polymerase exhibits low but noticeable activity during the reaction set-up. As a result non-specific priming events may lead to generation of unexpected amplification products during PCR. To avoid this, when using Taq polymerase the PCR reaction set-up should always be performed on ice.
Alternatively, use hot start PCR enzymes that have no activity at room temperature and are activated only at high temperatures during PCR cycling, e.g. TrueStart™ Hot Start Taq DNA Polymerase, Maxima® Hot Start Taq DNA Polymerase or PyroStart™ Fast PCR Master Mix (2X). In hot start PCR non-specific amplification is minimized and target yield is increased. PCR can be set-up at room temperature with hot start enzymes.

2.3. Excess Mg²⁺ concentration

If the Mg²⁺ concentration is too high, non-specific PCR products may appear. The recommended concentration range for optimizations is 1-4 mM.
For standard PCR with 0.2 mM dNTP and Fermentas Taq DNA Polymerase, a good starting point of MgCl₂ concentration is 1.5 mM (for Taq buffer with KCl) and 2.0 mM (for Taq buffer with (NH₄)₂SO₄). For DreamTaq™ DNA Polymerase or PCR Enzyme Mixes – we recommend a starting concentration of 1.5 mM MgCl₂. For Pfu DNA Polymerase – 2 mM Mg₂SO₄ and DreamTaq™ DNA Polymerase – 2 mM MgCl₂ is a good starting point.

2.4. Template amount too high

Optimal amounts of template DNA in a 50 µl reaction volume are in the 0.01-1 ng range for both plasmid and phage DNA, and in the 0.1-1 µg range for genomic DNA. Higher amounts of template increase the risk of generation of nonspecific PCR products.

2.5. Suboptimal thermal cycling conditions

The optimal annealing temperature normally is about 5°C lower than the primer-template melting temperature (Tm). The annealing temperature may be optimized stepwise in 1-2°C increments. If available, use a gradient cycler to optimize the annealing temperature of a specific PCR (±10°C).
The annealing temperature also has to be adjusted when additives that change the melting temperature of the primer-template duplex are used (e.g. glycerol, DMSO, formamide, betaine, TMANO (trimetylamine-N-oxide) or hydroxy-ectoine).

3. Sequence errors in PCR product

3.1. Low fidelity thermostabile DNA polymerase used in PCR

For downstream applications such as cloning or site-directed mutagenesis, high fidelity thermostabile DNA polymerases, such as Pfu DNA Polymerase or High Fidelity PCR Enzyme Mix are recommended.

3.2. Sub-optimal reaction conditions

Excess Mg²⁺ concentration. If the Mg²⁺ concentration is too high the fidelity of the PCR decreases. The recommended Mg²⁺ concentration range for PCR optimizations is 1-4 mM.
For standard PCR with 0.2 mM dNTP and Fermentas Taq DNA Polymerase, a good starting point of MgCl₂ concentration is 1.5 mM (for Taq buffer with KCl) and 2.0 mM (for Taq buffer with (NH₄)₂SO₄). For DreamTaq™ DNA Polymerase, 2 mM MgCl₂. For PCR Enzyme Mixes we recommend a starting concentration of 1.5 mM MgCl₂. For Pfu DNA Polymerase a good starting point is 2 mM MgSO₄.
Suboptimal template concentration. Optimal amounts of template DNA in the 50 µl reaction volume are in the 0.01-1 ng range for both plasmid and phage DNA, and in the 0.1-1 µg range for genomic DNA. Lower amounts of template reduce the accuracy of the amplification.
Imbalanced dNTP concentration. It is very important to have equal concentrations of all the nucleotides (dATP, dCTP, dGTP and dTTP) in the reaction. If the nucleotide concentrations are not balanced, the PCR error rate may dramatically increase. Fermentas dNTP Mixes contain either 2 mM or 10 mM, or 25 mM of each nucleotide. The concentrations of all four dNTPs are perfectly balanced to provide fidelity and to increase the yield of PCR products.

3.3. Exposed to UV PCR product

Use a long UV wavelength (360 nm) light-box when analyzing and excising PCR products from the agarose gel. When using a short-wavelength (254-312 nm) light-box, limit exposure to UV to several seconds. Keep the gel on a glass plate or on a plastic plate during illumination with UV. Alternatively, use dyes visible in ambient light to visualize PCR products in standard agarose gels (1, 2).
References

Rand, K.N., Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis and to Improve Cloning Efficiency, Elsevier Trends Journals Technical Tips, Online, T40022, 1996.
Adkins, S., Burmeister, M., Visualization of DNA in agarose gels and educational demonstrations, Anal Biochem., 240 (1), 17-23, 1996.

3.4. Sequence errors at PCR product termini

DNA sequence errors at the end of the PCR product can only be identified after subsequent cloning of the PCR product.

3.5. Faulty primer design

Avoid direct repeats in primers as multiple repeats may appear at the ends of the PCR product.

3.6. Low primer quality

As oligonucleotides are synthesized in the 3'=>5' direction near the 5'-end sequence inconsistencies may appear. Reorder primers from a reliable supplier.

3.7. Alteration of PCR product ends during cloning

Nuclease contamination. DNA nucleases present during extraction procedures or in the digestion or ligation reaction mixture may alter the ends of the PCR product.
DNA polymerase present in digestion reaction mixture. Remove active thermophilic DNA polymerases before digestion of a PCR product by spin column purification or phenol/chloroform extraction and subsequent ethanol precipitation. DNA polymerase may alter the ends of cleaved PCR products.
PCR product damaged by exposure to UV light. Use a long wavelength UV (360 nm) light-box when analyzing and excising PCR products from the agarose gel. When using a short-wavelength (254-312 nm) light-box, limit exposure to UV to several seconds. Keep the gel on a glass plate or on a plastic plate during illumination with UV. Alternatively, use dyes visible in ambient light to visualize PCR products in standard agarose gels (1, 2).

3.8. Sequencing error

To verify the reliability of sequencing results, sequence both DNA strands. Ensure sequencing primers are located in a distance of at least 20 nucleotides from the insertion site on cloning vector.
References

Rand, K.N., Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis and to Improve Cloning Efficiency, Elsevier Trends Journals Technical Tips, Online, T40022, 1996.
Adkins, S., Burmeister, M., Visualization of DNA in agarose gels and educational demonstrations, Anal Biochem., 240 (1), 17-23, 1996.

4. PCR/RT-PCR product in negative control

4.1. Cross-over contamination

PCR was contaminated by DNA or RNA present in the working environment.

4.2. Carry-over contamination

PCR contaminated by amplicons from previous reactions. If the same amplicon is to be generated multiple times, use carryover contamination control techniques. A common method used to avoid carry-over contamination is to incorporate dUTP into PCR products generated in the working environment followed by treatment with UDG (1).
Reference

Longo, M.C., et al., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions, Gene 93, 125-8, 1990.

4.3. Faulty primer design

DNA templates. Avoid direct repeats, self-complementarities and complementarities in between primer pairs, as primer multimers generate unexpected product.
RNA templates. To avoid amplification of genomic DNA, design PCR primers on exon-intron boundaries. Remove gDNA from RNA using DNase I, RNase-free.

4.4. RNA template contamination with genomic DNA

PCR product in the negative control denotes contamination with genomic DNA. Perform DNase I digestion prior reverse transcription.

5. Inefficient PCR cloning

5.1. Incorrect choice of polymerase

For generation of PCR products suitable for direct blunt-end cloning use a proofreading DNA polymerase such as Pfu DNA Polymerase.
For TA cloning use DreamTaq™ DNA Polymerase, Maxima® Hot Start Taq DNA Polymerase, TrueStart™ Hot Start Taq DNA Polymerase, Taq DNA Polymerase or other non-proofreading polymerases.
For efficient cloning with any DNA polymerase, use the CloneJET™ PCR Cloning Kit.

5.2. Faulty primer design.

When introducing restriction endonuclease sites into primers for subsequent digestion and cloning of a PCR product, refer to Table "Cleavage Efficiency Close to the Termini of PCR Fragments" to define the number of extra bases required for efficient cleavage by conventional Restriction Enzymes. For efficient digestion of PCR product follow the protocol.
For FastDigest® restriction enzymes follow the recommendations given in Table "Reaction Conditions for FastDigest® Restriction Enzymes" and in the protocol for fast digestion of PCR products.

5.3. Low primer quality

As oligonucleotides are synthesized in the 3'=>5' direction inconsistencies may appear in the 5' end. Reorder primers from a reliable supplier.

5.4. DNA polymerase is present in digestion reaction mixture

For cloning purposes remove active thermophilic DNA polymerase before digestion of a PCR product by spin column purification or phenol/chloroform extraction and subsequent ethanol precipitation. DNA polymerase may alter the ends of cleaved PCR products and reduce the ligation efficiency.

5.5. Restriction enzyme is sensitive to PCR mixture components

For efficient digestion of PCR product by conventional restriction enzymes follow the digestion protocol. For FastDigest® restriction enzymes follow the recommendations in the protocol for fast digestion of PCR products.
In case digestion of larger amounts of PCR products is desired scale up the above mentioned protocols according to the given recommendations. If digestion in smaller volumes is necessary, purify the PCR product by spin column or phenol/chloroform extraction and subsequent ethanol precipitation.

5.6. Final extension step is too short

This step can be prolonged to 20-30 minutes in the PCR cycling protocol to ensure a high efficiency of dA-tailing of PCR product, which can result in up to 3-4 fold higher numbers of recombinant clones.

5.7. Faulty primer design

The terminal transferase (3'-end extension) activity of Taq DNA polymerase exhibits template specificity with respect to the 3'-terminal nucleotide. Therefore, for efficient TA cloning of PCR products it is important to consider the 5'-end nucleotide of the primers. 5'-end nucleotides can be listed in the following order (according to dA-tailing efficiency): G> C >T >A (3).

5.8. Nuclease contamination

DNA nucleases present during extraction procedures or in the digestion or ligation reaction mixture may alter the ends of the PCR product.

- Troubleshooting Guide for PCR in PDF

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1. Low yield or no transformants

1.1. Low transformation efficiency of competent E.coli cells

Check transformation efficiency with 0.1 ng of a supercoiled vector DNA, e.g. pUC19 DNA. The competent cells should yield at least 1x10⁶ transformants per µg of supercoiled DNA, which corresponds to 100 colonies, when 0.1 ng of plasmid had been used for transformation.

1.2. Ligase and/or PEG were not removed prior to electroporation

When electroporation is used for transformation of blunt-end ligation reaction mixture, chloroform extraction instead of heat inactivation prior to electroporation is recommended.

1.3. Ligase was not heat inactivated

Heat inactivation of ligase improves the yield of transformants.

1.4. Excessive amount of ligation mixture used for transformation

Do not use more than 5 µl of ligation mixture for 50 µl of chemically competent cells and 1 µl for electrocompetent cells.

1.5. Cloned sequence is not tolerated by E.coli

Check the target sequence for strong E.coli promoters or other potentially toxic elements, as well as inverted repeats. In cases where the product of a cloned gene is toxic to the host, use promoters with a very low expression background or choose a low copy plasmid as cloning vehicle.

1.6. Excessive amount of ligase used for ligation

Do not use more ligase than it is recommended in the protocol relative to the vector and insert concentration. For easier manipulation Fermentas provides T4 DNA Ligase at 1 u/µl (#EL0015).

1.7. DNA contains contaminants

Ensure DNA is free of contaminants (e.g. excess salts, EDTA, proteins, phenol, etc.) that may inhibit ligation. Gel purify and/ or phenol/chloroform extract the vector and insert prior to ligation.

1.8. DNA was damaged by UV light during excision from the agarose gel

Use a long wavelength UV (360 nm) light-box when excising DNA from the agarose gel. When using a short-wavelength (254-312 nm) light-box, limit DNA exposure to UV to a few seconds. Keep the gel on a glass or plastic plate during UV illumination. Alternatively, use dyes visible in ambient light to visualize DNA in standard agarose gels (1, 2).
Another method to avoid exposure to UV is to load your sample in two or more lanes and then cut and stain only one lane with ethidium bromide after electrophoresis. Use this stained lane as a reference for excising the DNA from unstained lane that is not exposed to UV light.
References

Rand, K.N., Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis and to Improve Cloning Efficiency, Elsevier Trends Journals Technical Tips Online, T40022, 1996.
Adkins, S., Burmeister, M., Visualization of DNA in agarose gels and educational demonstrations, Anal. Biochem., 240 (1), 17-23, 1996.

1.9. Incorrect restriction enzyme chosen for DNA digestion

Incompatible vector and insert ends. Recheck the cloning strategy and choose restriction enzymes generating compatible overhangs for ligation.

1.10. Vector and insert are nonphosphorylated

If using dephosphorylated vectors, make sure the insert possesses phosphates. PCR products generally lack phosphate groups and need to be phosphorylated with T4 Polynucleotide Kinase prior to ligation. The CloneJET™ PCR Cloning Kit is compatible with both phosphorylated and dephosphorylated DNA fragments.

1.11. Inefficient blunting of DNA ends

Use the appropriate DNA blunting method for the type of DNA fragment ends.

1.12. Inefficient cleavage of PCR product

When introducing restriction enzyme sites into primers for subsequent digestion and cloning, refer to the Table "Cleavage Efficiency Close to the Termini of PCR Fragments" to define the number of extra bases required for efficient cleavage.
Prior to digestion remove the active thermophilic DNA polymerase from the PCR mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the ligation yield.
After digestion, gel-purify the PCR product to remove short DNA fragments, which compete with the insert in the ligation reaction.

1.13. Contaminated enzymes used for cloning

Use only the highest quality enzymes for cloning, e.g LO-tested enzymes and exclude any possibility of endo-, exo-nuclease and phosphatase contamination in enzyme preparations.

2. Empty vector (no insert)

2.1. Vector recircularization

Dephosphorylate the vector with FastAP™ Thermosensitive Alkaline Phosphatase, CIAP or SAP prior to ligation. Vector dephosphorylation is recommended in all cases, including cloning strategies where the vector ends are incompatible for recircularization. Ensure the phosphatase is completely inactivated or removed after dephosphorylation.

2.2. Incomplete cleavage of the vector

Check the cleavage efficiency on an agarose gel. If it is difficult to achieve complete cleavage, gel-purify the linear form of the vector using the DNA Gel Extraction Kit.

3. Incorrect constructs

3.1. Non-specific PCR product cloned

Gel-analyze the PCR product prior to ligation. If non-specific PCR products or primer-dimers were generated during the PCR reaction, gel-purify the target PCR product. Smaller DNA fragments present in the PCR mixture are ligated more efficiently with the cloning vector and out-compet the target PCR products.
Gel-purify the PCR product if the PCR template encodes beta-lactamase to avoid background colonies on LB-ampicillin agar. If the template and expected PCR product are of similar size, digest the template within the ampicillin resistance gene following the PCR reaction, e.g. with PdmI.

3.2. Truncated insert due to contaminating endo- or exonucleases

Use only the highest quality enzymes for cloning, e.g. LO-tested enzymes and exclude any possibility of endo-, exonuclease and phosphatase contamination in enzyme preparations.

4. Sequence errors in insert

4.1. Low fidelity DNA polymerase was used in PCR

If PCR product will be used for cloning it is always recommended to use high fidelity DNA polymerase with proofreading activity, such as Pfu DNA Polymerase.

4.2. DNA was damaged by UV light during the excision from agarose gel

Use a long wavelength UV (360 nm) light-box when excising DNA from the agarose gel. When a short-wavelength (254-312 nm) light-box is used, limit DNA exposure to UV to a few seconds. Keep the gel on a glass or on plastic plate during UV illumination. Alternatively, use dyes visible in ambient light to visualize DNA in standard agarose gels (1, 2).
References

Rand, K.N., Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis and to Improve Cloning Efficiency, Elsevier Trends Journals Technical Tips Online, T40022, 1996.
Adkins, S., Burmeister,M., Visualization of DNA in agarose gels and educational demonstrations, Anal. Biochem., 240 (1), 17-23, 1996.

4.3. Errors in PCR primers

If the cloned PCR product contains sequence errors or is missing 5' bases and the same error persits in more than one clone, re-order the PCR primers from a reliable supplier and repeat the procedure starting from the PCR step.

5. Colonies without plasmid

5.1. Insufficient amount of antibiotic in agar medium

Use 100 µg/ml of ampicillin in LB-ampicillin agar plates. Allow the LB medium to cool to 55°C before adding the antibiotic. Ampicillin is sensitive to light – long-term exposure to light can lead to low ampicillin concentration in plates.

5.2. Satellite colonies

Some fast growing E.coli strains (e.g. C600) degrade ampicillin faster, which leads to formation of smaller satellite colonies around transformants after >16 hours of incubation. Use shorter incubation times and do not use small satellite colonies for clone analysis.

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1. Low yield or no RNA transcript

1.1. Non-optimal reaction conditions

Conventional in vitro transcription reaction, using stand alone RNA polymerases should produce at least 10 µg of RNA transcript from 1 µg of template. For higher RNA yields (up to 200 µg), the TranscriptAid™ T7 High Yield Transcription Kit should be used.
Addition of Pyrophosphatase, Inorganic at 0.02-0.1 u (0.2-1 µl) per 20 µl of reaction volume may increase the yield of RNA by reducing the effect of reaction inhibition by pyrophosphates.

1.2. RNase contamination

Working environment, DNA template, reagents or electrophoresis systems may be contaminated with RNases.

Follow general recommendations for working with RNA.
Use RNase-free enzymes, nucleotides and DEPC-treated water.
Use RiboLock™ RNase Inhibitor to protect synthesized RNA from RNases.

Note

RiboLock™ RNase Inhibitor inhibits the activity of RNases A, B and C. It does not inhibit the following RNases: I, T1, T2, H, U1, U2 and CL3.
Do not use electrophoresis tanks which have been previously used for plasmid DNA miniprep analysis as they may be contaminated with RNases A or T1.

1.3. Insufficient yield of short transcript

High yields of short transcripts (mote than 100 bases) can be achieved by increasing the amount of template and extending the incubation time. Use 2 µg of template and prolong the reaction time to 4-8 hours.

1.4. DNA template of low purity or concentration

Evaluate your template in conjunction with a control template to determine if contaminants are inhibiting the reaction. If your template generates considerably lower RNA yields compared to the control template, modify the transcription reaction by mixing equal amounts of experimental template to the control template and adjusting the volume of DEPC-treated water. Evaluate the transcript on agarose gel:

Figure. Evaluation of mixing experiment results
C – control template
S – sample template
C/S1 – mixture of C and S: control reaction inhibited by sample template solution
C/S2 – mixture of C and S: control reaction not inhibited by sample template

If control reaction was inhibited by sample template (see Fig. Evaluation of mixing experiment results. C/S1), follow 1.5:

1.5. Reaction inhibitors in template DNA solution

Template DNA may contain residual SDS, EDTA, proteins, salts* and RNases. Repurify the template by phenol/chloroform extraction and ethanol precipitation. An A₂₆₀/₂₈₀ ratio of 1.8-2.0 should be observed. To remove EDTA and salts, wash the pellet with 70% cold ethanol.
* T7 and SP6 RNA Polymerases are inhibited ~50% by NaCl or KCl at concentrations above 150 mM (T3 RNA Polymerase at above 250 mM). Greater than 50% inhibition of the polymerases is observed with ammonium sulphate.

If control reaction is not inhibited by simple template (see Fig. Evaluation of mixing experiment results. C/S2), but low RNA yields are observed, follow 1.6:

1.6. Insufficient amount of template

Low amounts of template produce significantly lower RNA yields. The presence of RNA and chromosomal DNA in the DNA template preparation may interfere with UV absorbance readings and lead to misinterpretation of template DNA concentration. To accurately determine the concentration, size and integrity of the template, analyze the DNA concentration by UV absorbance and gel electrophoresis.

1.7. Incorrect reaction preparation

If the reaction is prepared on ice or in the incorrect order, the DNA may precipitate in the presence of spermidine in the reaction buffer. Water should always be added to the transcription reaction first.

2. Transcript is larger than expected

2.1. Incomplete cleavage of template plasmid DNA

Even small amounts of undigested circular DNA can produce large amounts of long transcripts. Check the template for complete digestion and, if required, additionally digest with the appropriate restriction enzyme. For faster and more efficient plasmid cleavage, use FastDigest® enzymes. If complete digestion is unachievable, gel-purify the digested template using DNA Gel Extraction Kit.

2.2. 3'-overhangs at DNA template ends

Avoid plasmid linearization with restriction enzymes that generate 3'-overhangs. Alternatively, blunt 3'-overhangs with T4 DNA Polymerase before use in transcription.

2.3. Aberrant migration of transcript

Due to secondary structures, RNA may run aberrantly on a native gel. On a denaturing gel, these transcripts normally migrate as single bands at the expected size.

3. Transcript smearing on denaturing agarose gel

3.1. RNase contamination in template DNA

During preparation, plasmid DNA templates are often contaminated with RNases. This can affect the length and yield of synthesized RNA, and is seen as a smear below the expected transcript length.
If using a commercial kit, such as the GeneJET™ Plasmid Miniprep Kit, omit the RNase A from plasmid preparation solutions and use DEPC-treated water for plasmid elution. If RNase A is premixed in the purification buffers, perform phenol/chloroform extraction after plasmid DNA linearization, then ethanol precipitate the DNA and dissolve in DEPC-treated water.

3.2. RNase contamination in working environment

Follow general recommendations for working with RNA.
Use RNase-free enzymes, nucleotides and water.
Use RiboLock™ RNase Inhibitor to protect synthesized RNA from RNases.

Note

RiboLock™ RNase Inhibitor inhibits the activity of RNases A, B and C. It does not inhibit the following RNases: I, T1, T2, H, U1, U2 and CL3.
Do not use electrophoresis tanks which have been previously used for plasmid DNA miniprep analysis as they may be contaminated with RNases A or T1.

4. Truncated transcript

4.1. RNA polymerase recognizes a termination signal in template DNA sequence

Try another RNA polymerase system or perform the transcription reaction at a lower temperature (e.g. 30°C). This may increase the length of transcript. However, RNA yield may be decreased at lower temperatures.

4.2. GC-rich DNA template (or template with high secondary structure)

For templates with secondary structure, incubating at 42°C or using a single-stranded binding (SSB) protein has been reported to improve yield and transcript length (1).
Reference

Aziz, R.B. and Soreq, H., Nucl. Acids Res., 18, 3418, 1990.

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1. Proteins poorly visualized

1.1. Insufficient staining

For Coomassie based staining, load ~0.5-5 µg of total protein per minigel well. 10 µg of total protein maybe required for lysates, 1-3 µg should be used to assay homogeneous protein. The staining sensitivity with PageBlue™ Protein Staining Solution is ~5 ng per band. Follow the protocol outlined in the manual.
For silver staining procedures load ~0.1-2 ng of total protein per minigel well. The staining sensitivity with PageSilver™ Silver Staining Kit is ~0.05-0.6 ng per band. Unstained protein ladder/marker can be visualized with PageBlue™ Protein Staining Solution, PageSilver™ Silver Staining Kit or other protein staining techniques.

1.2. Incorrect gel percentage

Linear gradient gels allow for adequate resolution of both small and large proteins. Homogeneous low percentage gels are recommended for analysis of large proteins and high percentage gels for analysis of small proteins. In high percentage gels (14-18%) large proteins (150-250 kDa) may not separate, while in low percentage gels (4-8%) small proteins will migrate with a tracking dye.
To choose the correct gel percentage for analysis of particular MW proteins, refer to Application Protocols for Protein Electrophoresis & Analysis (SDS-PAGE).

1.3. Excessive electrophoresis run time

Stop the electrophoresis run as soon as the tracking dye front reaches the bottom of the gel.
In low percentage gels (4-8%), small proteins (10-15 kDa) migrate with the tracking dye during electrophoresis and may be not visible. Use high percentage or gradient gels to resolve low molecular weight proteins.
To choose the right gel percentage for analysis of particular MW proteins, refer to General Recommendations for SDS-PAGE.

1.4. Loading recommendations not followed

Follow loading recommendation. Heat protein probes and Unstained Protein Molecular Weight Marker as described. Do not heat other Fermentas protein ladders/marker.

2. Smeared/diffused protein bands

2.1. Protein degradation by proteases

Use clean tips and vials when handling proteins. Use protease inhibitors when extracting proteins.
Store protein samples, ladders and markers at -20°C.

2.2. Stacking gel not used with the resolving gel

Placement of the stacking gel on top of the resolving gel is necessary to concentrate protein samples and to ensure accurate migration and separation into sharp bands.
Follow the gel preparation recommendations.

2.3. Improper sample preparation

To ensure proper migration during electrophoresis, protein samples should contain SDS, dithiothreitol (DTT) or 2-mercaptoethanol and must be heated prior to loading. Follow the recommendations for protein sample preparation and for protein ladders/markers.

2.4. Gel overloaded

For Coomassie based stains and Western blot applications use 0.5-5 µg of total protein per minigel well.
For silver staining procedures use 0.1-2 ng of total protein per minigel well and dilute Fermentas protein ladder/marker 50 times just prior to use.

3. Inaccurate sizing of proteins

3.1. Incorrect gel percentage

Use high percentage gels for analysis of small proteins and low percentage gels for analysis of large proteins. A gradient gel is ideal for precise determination of protein molecular weights. Refer to General Recommendations for SDS-PAGE to identify the correct percentage gel for a particular protein size.

3.2. Incorrect choice of ladder/marker for precise sizing

For precise determination of molecular weights, only unstained protein ladders/markers should be used. Prestained standards are recommended only for approximate protein sizing, as chromophores that are covalently coupled to the prestained proteins affect their mobility in various SDS-PAGE-buffer and gel systems. However, they are suitable for approximate molecular weight determination when calibrated against unstained standards in the same system.

3.3. Inaccurate protein sizing method

Always create a standard curve based on the mobility of protein standards after digitizing a gel image. The standard protein mobility data can be used to prepare a graph of the relationship between the molecular weight of standard proteins and their relative mobility (R_f). Usually the functional relationship is calculated according to the formula log (MW) = a + b x R_f, where a and b are constants determined by calibration with known standards. The MW of an unknown protein is calculated by substituting its R_f in the equation outlined above.
A new equation must be calculated for each gel, and data for several gels may be processed to create statistically robust results.

3.4. Improper sample preparation

To ensure proper migration during electrophoresis, protein samples must contain SDS, dithiothreitol (DTT) or 2-mercaptoethanol and must be heated prior to loading. Follow recommendations for protein sample preparation and for protein ladders/markers.

3.5. Excess salt concentration in the sample

High salt concentration in the sample will alter protein mobility. Remove excess salts by gel filtration.

3.6. Suboptimal electrophoresis conditions from those used for ladder/marker calibration

The apparent molecular weights of Fermentas prestained protein standards are calibrated in classical Tris-glycine-SDS Laemmli system. Each lot of prestained protein ladder/marker is calibrated against a precisely sized unstained protein ladder/marker in Tris-glycine gel and the calculated apparent molecular weights are reported in the product's Certificate of Analysis. However, the bands of the protein standard may have different mobilities in other electrophoresis buffer and gel systems.

3.7. Migration discrepancies due to protein modifications

Natural protein modifications such as; phosphorylation and glycosylation, may alter protein mobility. The molecular weights of modified proteins may or may not correspond to those of unmodified standard proteins of the same size.

4. Smiling, curved bands

4.1. Excessive voltage during electrophoresis run

Set the voltage to 250 V. Depending on a number of gels you run, use the appropriate power according the recommendations. Increase the power when dye front reaches the separating gel.

4.2. Insufficient buffer volume

Fill the electrophoresis tank (bottom and top reservoirs) with fresh 1X Tris-glycine-SDS buffer, make sure that the gel wells are completely covered with buffer.
Use cold buffer for electrophoresis.

4.3. Bubbles, physical particles in the gel

Mix and pour all gel preparation solutions carefully to avoid formation of bubbles. If physical particles are visible in solutions, remove them by filtration.

5. Suboptimal protein transfer

5.1. Improper Western transfer procedure

Follow the recommendations for Western blot transfer. For semi-dry Western transfers follow the protocol.
Make sure that buffer solutions completely cover the gel/membrane/paper sheets during all steps.
Use unstained gels for transfer, as stained proteins are transferred with lower efficiency.
Use prestained protein ladders and the DualColor™ Protein Loading Buffer Pack for electrophoresis as they allow for monitoring of the transfer efficiency.

5.2. Improper sample preparation for loading

To ensure proper migration during electrophoresis, protein samples must contain SDS, dithiothreitol (DTT) or 2-mercaptoethanol and must be heated prior to loading. Follow recommendations for protein sample preparation and for protein ladders/markers.

5.3. Low quality membrane used for transfer

Choose high quality PVDF membrane for Western blotting procedures. Low MW proteins are frequently transferred through nitrocellulose membranes and therefore may be not visible on the blot.

5.4. Errors SDS-PAGE procedure

Follow specific recommendations for protein electrophoresis.
Use prestained protein ladders as they allow for monitoring of electrophoresis and transfer efficiency.

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1. Low intensity of all or some of the DNA bands

1.1. Insufficient amount of ladder was loaded

Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1 mm gel lane width).

1.2. Insufficient or uneven staining

Following electrophoresis, visualize DNA by staining in ethidium bromide solution (final concentration 0.5 µg/ml) or SYBR® Green I.
Alternatively, if the DNA will not be used for cloning, add ethidium bromide to both the gel and electrophoresis buffer at a final 0.5 µg/ml concentration.
After alkaline agarose gel electrophoresis the gel should be immersed for 30 min in 300 ml 0.5 M Tris-HCl buffer, pH 7.5 and only later stained in a 0.5 µg/ml ethidium bromide solution for 30 min.
After denaturing polyacrylamide gel electrophoresis with urea, soak the gel for about 15 minutes in 1X TBE to remove the urea prior to staining. Stain the gel in 0.5 µg/ml ethidium bromide in 1X TBE solution for 15 min.
Make sure that the gel is immersed completely in the staining solution.

1.3. DNA run off the gel

Perform electrophoresis until the bromophenol blue dye passes 2/3 (orange G, 4/5) of the gel. Refer to General Recommendations for DNA Electrophoresis for migration of tracking dyes in different gels.
Make sure that the entire gel is immersed completely in the electrophoresis buffer during the run. Make sure that gel and apparatus are positioned horizontally during the run.

1.4. DNA diffusion in the gel

Avoid prolonged electrophoresis or excessive staining and destaining procedures as this may cause diffusion of smaller DNA fragments in the gel.
Avoid long term storage of the gel before taking a picture, as this may cause diffusion of DNA fragments and low band intensity.

1.5. DNA masking by electrophoresis tracking dyes

Do not exceed the amount of electrophoresis tracking dyes used for sample/ladder preparation. Use the loading dye solutions supplied with every Fermentas DNA ladder/marker, as these solutions contain equilibrated amount of tracking dyes which will not mask DNA under UV light.
Prepare DNA ladders and probes according to recommendations.

2. Smeared DNA bands

2.1. DNA degradation by nucleases

Use fresh electrophoresis buffers, freshly poured gels, nuclease free vials and tips to minimize nuclease contamination of DNA solutions.

2.2. Improper electrophoresis conditions

Prepare gels according to recommendations, always use the same electrophoresis buffer for both preparation of the gel and running buffer.
Make sure that the whole gel is immersed completely in the electrophoresis buffer during the run.
Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To increase the band sharpness, use a lower voltage for several minutes at the beginning of electrophoresis.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler™ or O'GeneRuler™ Express DNA ladders.
An excessively low voltage during the entire run may result in diffusion of bands during electrophoresis. Excessively high voltage may result in gel heating and DNA denaturation.
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (usually 5-8 V/cm, depending on the ladder) one has to:

measure the distance between electrodes (cathode and anode) – X, cm,
and multiply that X, cm value by the recommended voltage (Y, V/cm),
the result (X, cm x recommended Y, V/cm) is Z – recommended voltage to be applied.

2.3. Gel shift effect

DNA binding proteins, such as ligases, phosphatases or restriction enzymes may alter DNA migration on gels and cause the DNA to remain in the gel well or gel shifting.
Lambda DNA or other DNA with long complementary overhangs may anneal and migrate atypically.
To correct for the above mentioned effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load.

2.4. Excess DNA loaded

Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1 mm gel lane width). If possible apply same requirements for DNA quantities for the samples as well.

2.5. High salt concentration in the sample

Samples containing high concentrations of salts may result in smeared or shifted band patterns.
Ethanol precipitation and washing the pellet with ice cold 75% ethanol or spin column purification prior to resuspending the sample in water or TE buffer helps eliminate saltspresent in the sample.

2.6. Poorly formed (slanted) gel wells

When inserting the comb into the gel, make sure that it is vertical to the gel surface and stable during gel casting and its solidification.

3. Atypical banding pattern

3.1. Lambda DNA marker was not heated prior to loading

All DNA markers generated from Lambda DNA, as well as lambda DNA digestion products should be heated at 65°C for 5 min and chilled on ice before loading on the gel in order to completely denature the cohesive ends (the 12 nt cos site of lambda DNA) that may anneal and form additional bands.

3.2. Denatured DNA

Excessively high voltage may result in gel heating and DNA denaturation.
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (often 5-8 V/cm, depending on the ladder) one has to:

measure the distance between electrodes (cathode and anode) – X, cm,
and multiply that X, cm value by the recommended voltage (Y, V/cm),
the result (X, cm x recommended Y, V/cm) is Z – recommended voltage to be applied.

For non-denaturing electrophoresis use the loading dye solutions supplied with every Fermentas DNA ladder/marker, as these solutions do not contain denaturing agents.
Prepare DNA ladders and probes according to recommendations.
Do not heat them before loading. Heating is required only for lambda DNA markers.

3.3. Different loading conditions for the sample and the ladder DNA

Always use the same loading dye solution (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
If possible always load equal or very similar volumes of the sample DNA and the ladder/marker DNA. The sample can be diluted with 1X loading dye.

3.4. Improper electrophoresis conditions

Excessive electrophoresis run times or voltage may result in migration of small DNA fragments off of the gel. Very short or slow electrophoresis may result in incompletely resolved bands.
Run gels at 5-8 V/cm until the bromophenol blue passes 2/3 (orange G, 4/5) of the gel. Refer to General Recommendations for DNA Electrophoresis for migration of tracking dyes in different gels.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler™ or O'GeneRuler™ Express DNA ladders.

3.5. Incorrect gel percentage or running buffer used

TAE buffer is recommended for analysis of DNA fragments larger than 1500 bp and for supercoiled DNA. TBE buffer is used for DNA fragments smaller tha 1500 bp and for denaturing polyacrylamide gel electrophoresis. Large DNA fragments will not separate well in TBE buffer.
The correct gel percentage is important for optimal separation of the ladder DNA; prepare gels according to recommendations. When preparing agarose gels always adjust the volume of water to accommodate for evaporation during boiling. Otherwise, the gel percentage will be too high and result in bad separation of larger DNA bands.
Refer to General Recommendations for DNA Electrophoresis for the range of effective separation of DNA in different gels.
Ethidium bromide interferes with separation of large DNA fragments. Do not include ethidium bromide in the gel and run buffer when large DNA (more than 20 kb) or supercoiled DNA is analyzed. Stain the gel following electrophoresis in a 0.5 µg/ml ethidium bromide solution for 30 min.

3.6. Atypical migration due to different DNA sequence or structure

During high resolution electrophoresis DNA fragments of equal size can migrate differently due to differences in DNA sequences. AT rich DNA may migrate slower than an equivalent size GC rich DNA fragment. The sequences of Fermentas DNA ladders are chosen to allow for highly accurate DNA migration according to size, however, due to differencies in nucleotide sequence or the overall DNA structure, sample migration can sometimes slightly differ from ladder band migration.
DNA structures such as nicked, supercoiled or dimeric molecules will always show different mobility on gels compared to an equivalent DNA size standard. See the picture below for migration of plasmid DNA forms:

Figure. Migration of plasmid DNA forms

GeneRuler™ 1 kb DNA Ladder
Undigested plasmid pUC19 DNA (2,7 kb), forms:
- upper band (~4 kb) – dimeric plasmid,
- below, less visible (~3.5 kb) – nicked plasmid,
- lowest band (~1.9 kb) – supercoiled plasmid.
Linearized plasmid pUC19 (2,7 kb) – migrates according to its size.

High level DNA modifications such as methylation, labeling with biotin or large fluorescent molecules also result in slower migration compared to unmodified DNA of the same size.

3.7. Gel shift effect

The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel or cause the DNA to remain in the gel wells.
Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical migration pattern.
To eliminate these effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load.
High salt concentration in the sample may also cause gel shift effects, see 3.8.

3.8. High salt concentration in the sample

Samples with a high salt concentration may give smeared or shifted band patterns.
Ethanol precipitation and washing the pellet with ice cold 75% ethanol or spin column purification prior resuspending DNA in water or TE buffer, helps eliminate salt from the sample.

4. Curved DNA bands

4.1. Gel incompletely immersed in electrophoresis buffer

Electrophoresis buffer should completely cover the entire gel during sample loading and run.

4.2. Low sample volume

The sample or the ladder volume should be large enough to fill 1/3 of the total capacity of the well. Large wells should not be used with small sample volumes. If needed the sample volume can be adjusted with 1X loading dye.

4.3. Improper electrophoresis conditions

Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To minimize band curving, use a lower voltage for several minutes at the beginning of electrophoresis.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler™ or O'GeneRuler™ Express DNA ladders.
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (which is in many cases 5-8 V/cm, depending on the ladder) one has to:

measure the distance between electrodes (cathode and anode) – X, cm,
and multiply the X value by the recommended voltage (Y, V/cm),
the result (X x Y) is the recommended voltage to be applied.

4.4. Bubbles or physical particles in the gel wells or in the gel

Use pure water, clean flasks and clean equipment for preparation of gels.
Pour the gel slowly avoiding formation of bubbles. Bubbles can be removed with a pipette tip.

5. DNA remains in the gel

5.1. Poorly formed gel wells

Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately.
Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to loading the sample.

5.2. Excess DNA loaded

Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1mm gel lane width). If possible load the same quantity of the sample.

5.3. Contamination of the DNA sample

Make sure that your sample DNA solution does not contain any precipitate.

5.4. Gel shift effect

The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel and cause the DNA to remain in the gel wells.
Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical band migration pattern.
To eliminate these effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load.

6. Incorrect quantification data

6.1. Different loading conditions for the sample and the ladder DNA

Always use the same loading dye solution (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
If necessary, adjust the concentration of the sample to approximately equalize it with the amount of DNA in the nearest band.
If possible always load equal or very similar volumes of the sample DNA and the ladder/marker DNA. The sample can be diluted with 1X loading dye solution.

6.2. Incorrect ladder band chosen for quantification of the sample

Always compare the sample band with a similar sized ladder band.

6.3. Improper quantification method used

If possible, quantify by video-densitometry while subtracting the gel background as this method is more precise than a visual comparison of the bands.

6.4. Uneven staining of the gel and high background staining can also interfere with gel quantification results

Make sure that the gel is immersed completely in the staining solution.
Following electrophoresis, visualize DNA by staining in ethidium bromide solution (final concentration 0.5 µg/ml) or SYBR® Green I. Do not exceed the recommended concentration of the dye for staining.
Avoid prolonged staining for more than 30 min as this may result in high background.
If the gel is to be stained during the run, ensure that the ethidium bromide is included in both the gel and running buffer, otherwise the staining will be uneven.
After alkaline agarose gel electrophoresis the gel should be immersed for 30 min in 300 ml of 0.5 M Tris-HCl buffer, pH 7.5 and only later stained in a 0.5 µg/ml ethidium bromide solution for 30 min.
After denaturing polyacrylamide gel electrophoresis with urea, soak the gel for about 15min in 1X TBE to remove the urea prior to staining. Stain the gel in 0.5 µg/ml ethidium bromide in 1X TBE solution for 15 min.

6.5. DNA masking by electrophoresis tracking dyes

Do not exceed the recommended amount of electrophoresis tracking dyes used for sample/ladder preparation. Use the loading dye solutions supplied with every Fermentas DNA ladder/marker, as these solutions contain equilibrated amount of tracking dyes which will not mask DNA under UV light.
Prepare DNA ladders and probes according to the recommendations.

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1. RNA bands are not visible

1.1. Insufficient staining

Use the 2X RNA Loading Dye for both conventional RiboRuler™ RNA ladder and RNA sample preparation prior to electrophoresis. This solution includes ethidium bromide at a concentration sufficient to stain RNA on denaturing formaldehyde agarose gels. Ready-to-use RiboRuler™ RNA ladders are premixed with 2X RNA Loading Dye.
If RNA fragments are separated on native agarose gels, additional staining with ethidium bromide (final concentration 0.5 µg/ml) is recommended.
If RNA is separated on a denaturing glyoxal/DMSO agarose gel, stain the gel in ethidium bromide solution (final concentration 0.5 µg/ml) in 0.5 M ammonium acetate for 15-30 min after electrophoresis. Wash the gel in a fresh 0.5 M ammonium acetate solution for 15-30 min.
If RNA is separated on a denaturing polyacrylamide gel with urea, soak the gel for about 15 min in 1X TBE to remove the urea prior to staining. Stain the gel in 0.5 µg/ml ethidium bromide in 1XT BE solution for 15 min.

1.2. No staining

If you are using loading dye which does not contain ethidium bromide, add ethidium bromide to both the agarose gel and electrophoresis buffer at a final concentration of 0.5 µg/ml.
Alternatively, stain the gel after electrophoresis with ethidium bromide (0.5 µg/ml ethidium bromide) for 20 min, or SYBR® Green II (follow supplier recommendations).

1.3. Insufficient amount of ladder was loaded

Follow the recommendations for loading described in the certificate of analysis of the RiboRuler™ RNA ladders (0.25 µl per mm gel lane for conventional ladders; 0.5 µl per mm gel lane for ready-to-use ladders).

1.4. RNA degradation

Minimize exposure to UV light as this may cause RNA degradation/fading.
RNA, including the RiboRuler™ RNA ladders, is extremely sensitive to degradation by ribonucleases. The use of fresh electrophoresis buffers, freshly poured gels, DEPC-treated solutions and protective gloves is recommended.

1.5. RNA diffusion from the gel

Avoid prolonged electrophoresis or excessive staining and destaining procedures as this may cause diffusion of smaller RNA fragments from the gel.
Avoid long term storage of the gel prior to photo documentation, as this may cause diffusion of RNA fragments and band fading.

1.6. RNA has run off the gel

Stop electrophoresis after the bromophenol blue passes two thirds down the length of the gel. In most denaturing agarose gel systems, bromophenol blue migrates slightly faster than 5S rRNA and xylene cyanol FF migrates slightly slower than 18S rRNA.
Make sure that the electrophoresis tank is in a completely vertical position.

2. Smeared RNA bands

2.1. RNA degradation by nucleases

RNA, including the RiboRuler™ RNA ladders, is extremely sensitive to degradation by ribonucleases. The use of fresh electrophoresis buffers, freshly poured gels, DEPC-treated solutions and protective gloves is recommended.

2.2. Improper storage or use of RNA ladders

Store RiboRuler™ RNA ladders at -20°C for 6 months or at -70°C for 24 months. Thaw the ladders on ice.

2.3. Excessive gel depth or sample volume

Use thin (~0.5 cm) gels and avoid loading of large volumes in the gel lane.

2.4. Improper electrophoresis conditions

Ensure that there is enough electrophoresis buffer in the electrophoresis apparatus and that the gel is immersed completely.
Do not use an excessively high voltage for electrophoresis. Run agarose gels at 5 V/cm (polyacrylamide/urea gels at 8 V/cm). To increase the band sharpness, use a lower voltage for several minutes at the beginning of electrophoresis.
However, very low voltage during the entire run may result in band diffusion.

2.5. Excessive RNA ladder loaded onto the gel

Follow the recommendations for loading described in the certificate of analysis of the RiboRuler™ RNA ladders (0.25 µl per mm gel lane for conventional ladders; 0.5 µl per mm gel lane for ready-to-use ladders).

2.6. Incompletely immersed gel

Always ensure that there is enough electrophoresis buffer in the electrophoresis apparatus.

3. Atypical banding pattern

3.1. Inefficient denaturation of the ladder

All RiboRuler™ RNA ladders should be heated to 70°C for 10 min, chilled on ice for 3 min and briefly centrifuged before loading on the gel in order to completely denature the RNA. Sample RNA should be prepared the same way.

3.2. Sub-optimal gel preparation.

Older formaldehyde has an acidic pH which may cause extra RNA bands on the gel. Use only fresh formaldehyde for optimal results.

3.3. Different loading conditions for the sample and the ladder

Both ladder and sample RNA should be prepared with the same loading dye solution and loaded under the same conditions.
After electrophoresis of total RNA samples in the presence of ethidium bromide, the 28S and 18S human rRNA should be clearly visible under UV illumination. Fast-migrating bands composed of 5.8S RNA and 5S RNA may also be visible depending on the RNA purification procedure. The intensity of the 28S RNA should be approximately twice the intensity of the 18S RNA.
The 28S human rRNA band migrates at approximately 5000 b and the 18S human rRNA band migrates at approximately 1900 b.

3.4. Improper electrophoresis conditions

Excessively long electrophoresis runs may result in migration of small RNA fragments off the gel.
Very short electrophoresis runs may result in incompletely resolved bands.
Run agarose gels at 5 V/cm (polyacrylamide/urea gels at 8 V/cm) until the bromophenol blue passes 2/3 of the gel length.
TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used to resolve RNA fragments smaller than 1500 b and for denaturing polyacrylamide gel electrophoresis.
The correct gel percentage is important for optimal separation of the ladder RNA; take into account the following: RiboRuler™ High Range RNA Ladder can be loaded on:

native 0.8-1.5% agarose gel with TAE buffer
denaturing formaldehyde 0.8-1.5% agarose gel with MOPS buffer
denaturing glyoxal/DMSO 0.8-1.5% agarose gel with sodium phosphate buffer

RiboRuler™ Low Range RNA Ladder can be loaded on:

native 1.7-2.5% agarose gel with TBE buffer
denaturing formaldehyde 1.7-2.5% agarose gel with MOPS buffer
denaturing glyoxal/DMSO 1.7-2.5% agarose gel with sodium phosphate buffer
denaturing 4-8% polyacrylamide gel with TBE buffer

3.5. Sub-optimal ethidium bromide concentration in sample and ladder

The 2X RNA Loading Dye allows for RNA visualization without additional staining of denaturing agarose gels. Addition of extra ethidium bromide to the ladder or sample is not recommended and may result in RNA migration in the direction of the cathode.
If RNA fragments are separated on native agarose gels or on polyacrylamide/urea gels, additional staining with ethidium bromide after electrophoresis is recommended.

3.6. Incompletely immersed gel

Always ensure that there is enough electrophoresis buffer in the electrophoresis apparatus.

4. High background staining

4.1. Excessively high ethidium bromide concentration or prolonged staining

Use ethidium bromide at a final concentration of 0.5 µg/ml.
Avoid prolonged staining of the gels.

4.2. Insufficient gel destaining

If the gel is extensively stained with ethidium bromide, additional destaining in water is needed to remove background staining.
Wash glyoxal/DMSO agarose gels after staining in a fresh 0.5 M ammonium acetate solution for 15-30 min.

5. Uneven staining of the gel

5.1. Improper gel staining conditions

Ethidium bromide migrates in the opposite direction of the RNA during electrophoresis. Therefore, if ethidium bromide is only added to the agarose gel and not to the electrophoresis buffer, it may result in uneven RNA fragment staining.
When 2X RNA Loading Dye is used for both conventional RiboRuler™ RNA ladders and RNA sample preparation prior to electrophoresis, additional staining is not required as the loading dye includes sufficient ethidium bromide to stain RNA on denaturing formaldehyde agarose gels.
For native agarose gels, ethidium bromide (0.5 µg/ml) should be added to both the electrophoresis buffer and the agarose. This ensures an even distribution of ethidium bromide during electrophoresis so that the intensity of the bands upon exposure to UV light will be proportional to the quantity of RNA present.

5.2. Incompletely immersed gel

Always ensure that there is enough electrophoresis buffer in the electrophoresis apparatus or enough of the staining solution during the staining so that the gel is always immersed completely.

6. RNA remains in the gel well

6.1. Poorly formed gel wells

Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately.
Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to loading the sample.

6.2. Large quantity of RNA loaded into the gel

Follow the recommendations for loading described in the certificate of analysis of the RiboRuler™ RNA ladders (0.25 µl per mm gel lane for conventional ladders; 0.5 µl per mm gel lane for ready-to-use ladders).

6.3. Contamination of the RNA sample

Make sure that your sample RNA solution does not contain any precipitate.

7. Incorrect quantification data

7.1. Impure RNA

Free NTPs and truncated transcripts remaining in the sample after in vitro transcription can interfere with spectrophotometrical measurements and lead to inaccurate quantification of sample RNA.
RiboRuler™ RNA ladders are produced from chromatography-purified RNA transcripts and are free of any NTPs and truncated transcripts. Therefore the gel quantification data is compatible with the spectrophotometrical measurements of RiboRuler™ RNA ladders.

7.2. Incorrect RiboRuler™ band chosen for quantification of the sample

Always compare the sample band with a similar size ladder band.

7.3. Different loading conditions for the ladder and samples

Both sample and ladder RNA should be loaded under the same conditions.
Use the supplied 2X RNA Loading Dye for the sample and ladder.
Load equal volumes of sample RNA and ladder RNA. The required volume of sample RNA can be obtained by diluting with a mixture (1:1) of DEPC-treated Water and 2X RNA Loading Dye.

7.4. Improper quantification method used

If possible, quantify by video-densitometry measurements while subtracting the gel background as this method is more precise than a visual comparison of the bands.

7.5. Uneven staining of the gel and high background staining

Uneven staining of the gel and high background staining can also interfere with gel quantification results (see Problem 4 and 5 above).

- Troubleshooting Guide for RNA Electrophoresis in PDF

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1. Low transfection efficiency

1.1. Inefficient complex formation

Always vortex the mixture immediately after the addition of the reagent to DNA.

1.2. Suboptimal reagent/DNA ratio

Optimize the quantity of transfection reagent added to the fixed amount of DNA.

1.3. Suboptimal quantity of DNA

Optimize the amount of DNA used for transfection. Keep the transfection reagent/DNA ratio constant.

1.4. Poor polyplex/cell surface contact

Centrifuge the culture plates, if it will not harm the cells. Caution – centrifugation may harm some primary cells.

1.5. Poor DNA quality

Use high quality DNA with an A₂₆₀/A₂₈₀ ratio greater than 1.8.

1.6. Suboptimal cell confluency

Optimize cell plating conditions. Ensure that adhered cells are 50-70% confluent at the time of transfection. Ensure that suspension cells are in logarithmic growth phase at the time of transfection.

1.7. Mycoplasma contamination

Mycoplasma infection in cell culture often results in poor and/or non-reproducible transfection. Regularly check your cells for mycoplasma infection.

2. High cellular toxicity

2.1. Toxic transgene

Verify if the expressed transgene is toxic.

2.2. Suboptimal incubation conditions

Reduce incubation time of the polyplexes with the cells. Replace the transfection mixture 3-6 hours later with fresh growth medium.

2.3. Suboptimal quantity of DNA

Reduce the quantity of DNA used for transfection.

2.4. Cell density is too low

Increase the plating density of cells used for transfection.

- Troubleshooting Guide for Transfection in PDF

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