Taq DNA Polymerase is a recombinant enzyme expressed and purified by Escherichia coli. The gene is derived from the Thermos aquaticus polymerase. The protein has a molecular weight of 94 kDa and exhibits 5 '→ 3' DNA polymerase activity and 5 '→ 3' exonase activity. There is no 3 '→ 5' exonase activity, and the enzyme elongation rate is 2 kb/min. It can amplify fragments up to 5 kb in length. The amplified PCR product has an "A" base attached to its 3 'end, making it suitable for direct T/A cloning. This product has the characteristics of fast extension speed and high amplification efficiency, and is mainly suitable for experiments such as PCR amplification of DNA fragments and DNA sequence determination.
Component :500U 2500U 10000U
Taq DNA Polymerase (5U/ μ L) 100 μ L 5 × one hundred μ L 2 × 1mL
ten × PCR Buffer 1.8mL 5 × 1.8mL 8 × 5mL
Storage: -20 ℃
Note:
10% of this product × The PCR buffer contains 15mM magnesium ions.
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 (50 μ L) :
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
Taq DNA Polymerase 0.25-0.5 μ L 1.25-2.5U/50 μ L
DdH2O up to 50 μ L
Note:
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 cycles
