Bes Taq DNA Polymerase
Bes Taq DNA Polymerase exhibits 5 '→ 3' DNA polymerase activity and 5 '→ 3' exonuclease activity. Under ordinary PCR conditions, compared with Taq DNA Polymerase, it has excellent performance of high amplification efficiency and low mismatch rate, as well as the high amplification efficiency of Taq DNA Polymerase and the high fidelity of Pfu DNA Polymerase. The PCR product amplified with this product has an "A" base at the 3 'end, so it can be directly used for T/A cloning. This product is suitable for routine PCR reactions and gene cloning reactions that require fidelity.
Component 500U
Be Taq DNA Polymerase (5U/ μ L) 100 μ L
ten × PCR Buffer 1.8mL
Storage: -20 ℃, avoid repeated freeze-thaw.
Using activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxynucleotides into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.
After multiple column purification, the purity was detected by SDS-PAGE to be over 99%;
No exogenous nuclease activity was detected;
PCR method for detecting non host residual DNA;
Effectively amplifying single copy genes in the human genome;
Store at room temperature for one month without significant changes in activity.
The following example is a PCR reaction system and reaction conditions for amplifying a 1kb fragment using human genome DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size.
PCR reaction system
Component dosage and final concentration
ten × PCR Buffer 5 μ L 1 ×
DNTP Mix, 10mM each 1 μ L 200 μ M each
Forward Primer, 10 μ M 2 μ L 0.4 μ M
Reverse Primer, 10 μ M 2 μ L 0.4 μ M
Template DNA<0.5 μ G<0.5 μ G/50 μ L
Be Taq DNA Polymerase (5U/ μ L) 0.25~0.5 μ L 1.25-2.5U/50 μ L
DdH2O to 50 μ L
Attention: The primer concentration should be between 0.1 and 1.0 at the final concentration μ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the concentration of primers can be reduced to optimize the reaction system.
PCR reaction conditions
Step Temperature Time Cycles
Pre denaturation at 94 ℃ for 2 minutes 1
Denaturation at 94 ℃ for 30s, 25-35
Annealing at 55~65 ℃ for 30 seconds
Extension 72 ℃ for 30s
Final extension 72 ℃ for 2 minutes 1
Attention:
In general experiments, the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, which cannot be obtained
