Random Hexamer Primer is a mixture of single-stranded random hexanucleotides with 5'- and 3'-hydroxyl ends. The primer is supplied as a ready-to-use, 20X concentrated aqueous solution.
Quality Control
Functionally tested in first strand cDNA synthesis.
Radioactive Random-primed DNA Labeling with Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment
Prepare the following reaction mixture:
DNA (aqueous solution)
10 µl (100 ng)
10X reaction buffer for Klenow Fragment, exo- or 10X Bsm buffer
5 µl
6.0 A260units/ml (100 µM) Random Hexamer Primer
12.5 µl
Water, nuclease-free
to 40 µl
Total volume
40 µl
Incubate the mixture in a boiling water bath for 5-10 minutes and then chill on ice.
Add:
3 dNTP Mix, 0.33 mM each (without a labeled dNTP)
3 µl (0.02 mM final concentration)
[alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)
1.85 MBq (50 µCi)
Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment
1 µl (5 u) 1 µl (8 u)
Water, nuclease-free
to 50 µl
Total volume
50 µl
Incubate the reaction mixture for 10 minutes at 37°C.
Add 4 µl 0.25 mM dNTP mix and incubate at 37°C for 5 minutes.
Add 1 µl 0.5 M EDTA, pH 8.0 to stop the reaction.
Remove 1 µl of the reaction mixture and determine the percentage of label incorporated.
Purify by using Sephadex G-50 or Bio-Gel P-60.
Non-radioactive Random-primed DNA Labeling with Klenow Fragment, exo-
Prepare the following reaction mixture:
DNA template
10 µl (100 ng – 1 µg)
10X reaction buffer for Klenow Fragment, exo-
5 µl
6.0 A260units/ml (100 µM) Random Hexamer Primer
12.5 µl
Water, nuclease-free
to 39 µl
Total volume
39 µl
Incubate the mixture in a boiling water bath for 5-10 minutes and then chill on ice.
Add:
3 dNTP Mix, 1 mM each (without the dTTP)
5 µl (0.1 mM final concentration)
dTTP
3.25 µl (0.065 mM final conc.)
Biotin-11-dUTP*, 1 mM
1.75 µl
Klenow Fragment, exo-
1 µl (5 u)
Total volume
50 µl
* Fluorescein-12-dUTP, DIG-dUTP or Aminoallyl-dUTP can also be used with the same protocol.
Incubate the reaction mixture at 37°C for 1 hour.
Add 1 µl 0.5 M EDTA, pH 8.0 to stop the reaction.
Remove 1 µl of the reaction mixture and determine the percentage of label incorporated.
Optionally, purify by using Sephadex G-50 or Bio-Gel P-60.
Components of the Reaction Mixture
Template RNA Total cellular RNA isolated by standard methods can be successfully used with Fermentas reverse transcriptases or the kits for first strand cDNA synthesis. The purified RNA has to be free of salts, metal ions, ethanol and phenol to avoid inhibition during reverse transcription reaction. Template RNA for RT-PCR has to be free of DNA contamination. It is recommended to use DNase I, RNase-free, to remove trace amounts of DNA from RNA preparations. Always perform a control RT PCR reaction with a RNA template that has not been transcribed with reverse transcriptase. Primers Synthesis of first strand cDNA can be primed with either oligo(dT)18, random primers or gene specific primers. Oligo(dT)18 primers cDNA synthesis from the poly(A) tail present at the 3'-end of eukaryotic mRNA. Random primers, e.g. hexamers, initiate cDNA synthesis from all RNA species (rRNA and mRNA) present in total RNA samples. This results in a greater complexity of the resulting cDNA than using just the oligo(dT)18 primer and may reduce sensitivity and/or specificity of subsequent PCR reaction. However, there are situations where random primers are preferred, such as cDNA synthesis using eukaryotic mRNAs without a poly(A) tail, or cDNA synthesis using a poly(A)-enriched RNA sample as well as RT-PCR of 5' regions of long mRNAs. Gene-specific primers provide the greatest specificity of cDNA synthesis; such primers must be obtained by the user. Enzymes Fermentas offers both wildtype and genetically engineered versions of the Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase and a recombinant AMV Reverse Transcriptase. All enzymes are suitable for the synthesis of full-length first strand cDNA, but they differ in reaction temperatures, amounts of RNA transcribed, sensitivity and RNaseH activity. See table below for the reaction conditions recommended for each of the enzyme. Enzyme units and RNA amounts are provided for 20 µl of RT reaction volume:
Reverse transcriptase
Reaction tmp.
Active up to
Reading length
RHase H activity
Inactivation
Units
Total RNA
poly(A) RNA
Maxima® RT
50-55°C
60°C
20kb
+
85°C, 5min
200
1pg-5µg
0.1pg-500ng
RevertAid™ Premium RT
50-55°C
60°C
20kb
–
85°C, 5min
200
1pg-5µg
0.1pg-500ng
RevertAid™ H Minus RT
42-45°C
55°C
13 kb
–
70°C, 10min
200
0.1ng-5µg
10pg-500ng
RevertAid™ RT
42°C
50°C
13kb
+
70°C, 10min
200
0.1ng-5µg
10pg-500ng
M-MuLV RT
37°C
37°C
9kb
+
70°C, 10min
40
100ng-5µg
10-500ng
AMV RT
45-60°C
60°C
13kb
++
85°C, 5min
10
10ng-5µg
1-100ng
First Strand cDNA Synthesis, an example (reaction set up)
This protocol is provided for first strand cDNA synthesis using RevertAid™ H Minus Reverse Transcriptase. The table below indicates reaction conditions recommended for each RT. Enzyme units and RNA amounts are provided for 20 µl of RT reaction volume:
Reverse transcriptase
Reaction tmp.
Active up to
Reading length
RHase H activity
Inactivation
Units
Total RNA
poly(A) RNA
Maxima® RT
50-55°C
60°C
20kb
+
85°C, 5min
200
1pg-5µg
0.1pg-500ng
RevertAid™ Premium RT
50-55°C
60°C
20kb
–
85°C, 5min
200
1pg-5µg
0.1pg-500ng
RevertAid™ H Minus RT
42-45°C
55°C
13 kb
–
70°C, 10min
200
0.1ng-5µg
10pg-500ng
RevertAid™ RT
42°C
50°C
13kb
+
70°C, 10min
200
0.1ng-5µg
10pg-500ng
M-MuLV RT
37°C
37°C
9kb
+
70°C, 10min
40
100ng-5µg
10-500ng
AMV RT
45-60°C
60°C
13kb
++
85°C, 5min
10
10ng-5µg
1-100ng
Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors and contamination, prepare a RT master mix by adding all reaction components except RNA into one vial. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Sample template RNA into individual tubes and keep on ice. Aliquote the prepared master mix into tubes with RNA. Mix and briefly centrifuge all components after thawing, keep on ice.
Add into sterile, nuclease-free tube on ice in the indicated order:
Template RNA
total RNA or poly(A) RNA or specific RNA
10 ng – 5 µg 1-500 ng 0.01 pg – 0.5 µg
Primer
Oligo(dT)18 or Random hexamer or Gene-specific
0.5 µg (100 pmol) 0.2 µg (100 pmol) 15-20 pmol
DEPC-treated Water
to 12.5 µl
Total volume
12.5 µl
Optional: If RNA template is GC rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min, chill on ice, briefly centrifuge and place on ice.
Add the following components in the indicated order:
5X reaction buffer
4 µl
RiboLock™ RNase Inhibitor
0.5 µl (20 u)
dNTP Mix, 10 mM each
2 µl (1 mM final concentration)
RevertAid™ H Minus Reverse Transcriptase
1 µl (200 u)
Total volume
20 µl
Mix gently and centrifuge briefly.
If oligo(dT)18 primer or gene-specific primer is used, incubate 60 min at 42°C. If random hexamer primers are used, incubate 10 min at 25°C followed by 60 min at 42°C. For transcription of GC rich RNA reaction temperature can be increased to 45°C.
Terminate the reaction by heating at 70°C for 10 min.
Note
The reverse transcription reaction product can be directly used in PCR or stored at -20°C.
Use 2 µl of the reaction mix to perform PCR in 50 µl of reaction volume.
- Worldwide
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- Store at -20°C
