- Products - All - PCR, qPCR, RT-PCR & dNTPs - DreamTaq™ Green PCR Master Mix (2X)

Features

Convenient – DreamTaq™ Green DNA Polymerase in a ready-to-use mix.
Save time – go directly from PCR to gel electrophoresis.
High yields of PCR products with minimal optimization.
Higher sensitivity compared to conventional Taq DNA polymerase.
Amplification of long targets up to 6 kb from genomic DNA and up to 20 kb from viral DNA.
Robust amplification of difficult templates.

Applications

Routine PCR amplification of DNA fragments up to 6 kb from genomic DNA and up to 20 kb from viral DNA.
RT-PCR.
Genotyping.
Generation of PCR products for TA cloning.

Description

DreamTaq™ Green PCR Master Mix (2X) is a ready-to-use solution containing DreamTaq™ DNA Polymerase, optimized DreamTaq™ Green buffer, MgCl₂ and dNTPs. The master mix is supplemented with two tracking dyes and a density reagent that allows for direct loading of PCR products on a gel. The dyes in the master mix do not interfere with PCR performance and are compatible with downstream applications such as automated fluorescent DNA sequencing, ligation and restriction digestion.

The master mix retains all features of DreamTaq™ DNA Polymerase. It is capable of robust amplification of up to 6 kb from genomic DNA and up to 20 kb from viral DNA.

For applications that require PCR product analysis by absorbance or fluorescence excitation, we recommend using the colorless DreamTaq™ PCR Master Mix (2X).

Quality Control

The absence of endo-, exodeoxyribonucleases and ribonucleases is confirmed by appropriate quality tests. Functionally tested in amplification of a single-copy gene from human genomic DNA and for amplification of 20 kb lambda DNA fragment.

Composition

DreamTaq™ DNA Polymerase is supplied in 2X DreamTaq™ Green buffer, dATP, dCTP, dGTP and dTTP 0.4 mM each and 4 mM MgCl₂.
DreamTaq™ Green buffer is a proprietary formulation optimized for robust performance in PCR. In addition, it contains a density reagent and two dyes for monitoring electrophoresis progress: the blue dye migrates with 3-5 kb DNA fragments in a 1% agarose gel and the yellow dye migrates faster than 10 bp DNA fragments in 1% agarose gel. The dyes have absorption peaks at 424 nm and 615 nm.

Figure 1. Robust amplification of human genomic DNA with DreamTaq™ Green PCR Master Mix.
A 2.5 kb tPA gene fragment was amplified from100 ng of human genomic DNA in a 50 µl PCR mixture using DreamTaq™ DNA polymerase in stand-alone and Master Mix formats.
M – GeneRuler™ Express DNA Ladder
1, 2 – PCR product generated with 1.25 u DreamTaq™ Green DNA Polymerase, DreamTaq™ Green Buffer and dNTPs
3, 4 – PCR product generated with DreamTaq™ Green PCR Master Mix (2X) (stored at -20°C for 7 months)
5, 6 – PCR product generated with DreamTaq™ Green PCR Master Mix (2X) (after 25 freeze-thaw cycles)
7, 8 – PCR product generated with 1.25 u of DreamTaq™ DNA Polymerase, DreamTaq™ Buffer and dNTPs
9, 10 – PCR product generated with DreamTaq™ PCR Master Mix (2X) (stored at -20°C for 7 months)
11, 12 – PCR product generated with DreamTaq™ PCR Master Mix (2X) (after 25 freeze-thaw cycles)

Patents, Licenses, Trademarks

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Analysis of Recombinant Clones

Analyze 4-6 white colonies for the presence and orientation of the DNA insert using one of the following methods.
Restriction analysis. Isolate plasmid DNA from an overnight bacterial culture using a convenient plasmid miniprep method such as GeneJET™ Plasmid Miniprep Kit. Use FastDigest® restriction enzymes to digest DNA from recombinant clones in just 5 min.
Sequencing. Isolate plasmid DNA from an overnight bacterial culture using a reliable plasmid miniprep method such as GeneJET™ Plasmid Miniprep Kit. Sequence the insert using appropriate sequencing primers.
Colony PCR. Use the following protocol for colony screening by PCR.

Prepare enough PCR master mix for the number of colonies analyzed plus one extra. For each 20 µl of reaction, mix the following reagents:

Component	Taq DNA Polymerase or DreamTaq™ DNA Polymerase	PCR Master Mix (2X)
*10X Taq* buffer**	2 µl	-
dNTP Mix, 2mM each	2 µl	-
25 mM MgCl₂	1.2 µl	-
M13/pUC Reverse Sequencing primer (#SO101)	0.6 µl (10 µM)	0.6 µl (10 µM)
M13/pUC Forward Sequencing primer (#SO100)	0.6 µl (10 µM)	0.6 µl (10 µM)
Taq DNA polymerase	0.1 µl (0.5 u)	-
PCR Master Mix (2X)	-	10 µl
Water, nuclease-free	to 20 µl	to 20 µl
Total volume	20 µl	20 µl

Mix thoroughly, spin briefly and aliquot 20 µl of the mix into the PCR tubes on ice.
Pick up an individual colony with a sterile pipette tip and resuspend it in 20 µl of the PCR master mix. Make a short strike with the same tip over culture plate to save the clone for repropagation.
Perform PCR: 95°C, 3 min; 94°C, 30 s, 45°C*, 30 s, 72°C 1 min/kb; 30 cycles.
Analyze on a gel for the presence of the PCR product of the expected length.
* Depends on primer pair used (Tm-5).

In vivo DNA Transfection using TurboFect™ in vivo Transfection Reagent

Reagents to be Supplied by the User: sterile solution of 5% glucose (w/v).
Protocol

Dilute 50 µg of DNA in 400 µl of a sterile 5% glucose solution. Vortex gently and centrifuge briefly.
Add 6 μl of TurboFect™ in vivo Transfection Reagent and mix the solution by pipetting.
Incubate for 15-20 min at room temperature.
Perform injections.
Monitor gene expression with the method most suitable for your studies.

Note

The A₂₆₀/A₂₈₀ ratio should be at least 1.8 for purified DNA. It is important to use endotoxin-free DNA (less than 0.1EU/1 µg DNA).
The amount of DNA and maximum injection volume depend on the experimental animal and the route of administration (see Tables 1 below) as well as on the targeted tissue or organ and on the expression vector.

Table 1. Suggested amount of DNA and maximum injection volume.

Animal	Route of injection	Suggested amount of DNA, µg	Maximum injection volume, µl	Reference
Adult mouse	intravenous injection	25-125	400-600	1, 6, 7, 9
Adult mouse	brain injection	2.5	5	5
Newborn mouse	brain injection	1	2	5
Nude mouse	intravenous injection	50	200	8
Nude mouse	subcutaneous tumor injection	10	100	8
Adult rabbit	tracheal injection	300-700	300-700	2, 4
Newborn rabbit	tracheal injection	300	300	4
Adult rat	brain injection	0.5	2	12
Tadpole	brain injection	0.5-1	1	10
Pekin Duck*	intravenous injection	400**	2000	3

Note

* 10 day old.
** 400 µg of fluorescein-labeled antisense oligodeoxynucleotides.
Table 2. Scale-up ratios

Amount of DNA, µg	Volume of TurboFect™ in vivo Transfection Reagent, µl
Amount of DNA, µg	recommended	range
1	0.12	0.1-0.16
5	0.6	0.5-0.8
10	1.2	1-1.6
50	6	5-8

References

Bragonzi, A., et al., Conese M., Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs, Gene Ther., Dec, 6(12), 1995-2004,1999.
Ferrari, S., et al., Polyethylenimine shows properties of interest for cystic fibrosis gene therapy, Biochim Biophys Acta, Oct 28, 1447(2-3), 219-25, 1999.
Chemin, I., et al., Liver-directed gene transfer: a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo, J. Viral Hepat, Nov, 5(6), 369-75, 1998.
Ferrari, S., et al., ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo, Gene Ther., Oct, 4(10), 1100-6, 1997.
Goula, D., et al., Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system, Gene Ther, May, 5(5), 712-7, 1998.
Goula, D., et al., Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes, Gene Ther., Mar, 7(6), 499-504, 2000.
Goula, D., et al., Polyethylenimine-based intravenous delivery of transgenes to mouse lung, Gene Ther., Sep, 5(9), 1291-5, 1998.
Coll, J.L., et al., In vivo delivery to tumors of DNA complexed with linear polyethylenimine, Hum Gene Ther., Jul 1, 10(10), 1659-66, 1999.
Zou, S.M., et al., Systemic linear polyethylenimine (L-PEI)- mediated gene delivery in the mouse, J. Gene Med, Mar-Apr, 2(2), 128-34, 2000.
Ouatas, T., et al., T3-dependent physiological regulation of transcription in the Xenopus tadpole brain studied polyethylenimine based in vivo gene transfer, Int J Dev Biol., Nov;42(8), 1159-64, 1998.
Boussif, O., et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc Natl Acad Sci U S A, Aug 1, 92(16), 7297-301, 1995.
Fabre, V., et al., Homeostatic regulation of serotonergic function by the serotonin transporter as revealed by nonviral gene transfer, J Neurosci., Jul 1, 20(13), 5065-75, 2000.