I want to add dATP on the ends of to my PCR product (Phusion, Thermo). My question is if it's possible with use of DreamTaq polymerase (Thermo)? I find informations about Taq polymerases but can not find out if i can use this polymerase to do that (a-tailing, not PCR itself, i got insert from PCR with Phusion polymerase).
Thx for help, cheers
The information sheet at
Thermo Scientific DreamTaq DNA Polymerase (5 U/ L) 5μ/μL, 500U:Molecular | Fisher Scientific
confirms that dream taq does crete a 3' A overhang.
Description
DreamTaq DNA Polymerase is an enhanced Taq DNA polymerase optimized for all standard PCR applications. It ensures higher sensitivity, longer PCR products and higher yields compared to conventional Taq DNA polymerase. DreamTaq DNA Polymerase uses the same reaction set-up and cycling conditions as conventional Taq DNA polymerase. Extensive optimization of reaction conditions is not required. It is supplied with optimized DreamTaq buffer, which includes 20 mM MgCl2.
DreamTaq PCR Master Mix (2X) is a ready-to-use solution containing DreamTaq DNA Polymerase, optimized DreamTaq buffer, MgCl2, and dNTPs. This pre-mixed formulation saves time and reduces contamination due to the fewer pipetting steps required for PCR set up. 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.
DreamTaq DNA Polymerase ensures higher sensitivity, longer PCR products and higher yields compared to conventional Taq DNA polymerase. DreamTaq DNA Polymerase uses the same reaction set-up and cycling conditions as conventional Taq DNA polymerase. Extensive optimization of reaction conditions is not required. It is supplied with optimized DreamTaq buffer, which includes 20 mM MgCl2. DreamTaq DNA Polymerase generates PCR products with 3'-dA overhangs. It does not incorporate dUTP.
- Robust amplification with minimal optimization
- High yields of PCR products
- Higher sensitivity compared to conventional Taq DNA polymerase
- Amplification of long targets up to 6kb from genomic DNA and up to 20kb from viral DNA
- Generates 3'-dA overhangs
- Incorporates modified nucleotides
It already has Mg in the buffer Hafida Ayada but if you are amplifying dna with pcr inhibitors in it ( common in plant and soil dna samples) then you may want to add more Mg as some inhibitors spoil the pcr by removing Mg from the reaction mix
I have a pair of primers with the following Tms:
ITS1F:
Tm (1M Na+) = 66
Tm (50mM Na+) = 44 I
TS4:
Tm (1M Na+) = 66
Tm (50mM Na+) = 45
which Tm should I choose to perform a PCR
I have a pair of primers with the following Tms:
ITS1F:
Tm (1M Na+) = 66
Tm (50mM Na+) = 44
ITS4:
Tm (1M Na+) = 66
Tm (50mM Na+) = 45
which Tm should I choose to perform a PCR
50mM salt is closest to pcr conditions so you should use the Tm at 50mM in your pcr. You may need to run the pcr at a lower annealing temperature than 44 to ensure that the primers anneal but running a temperature gradient of annealing temperatures is always a good idea
thank you for this answer
I carried out a PCR with Tm =44 but there is no amplification
- Badges
- Science method