Manual

Product Number: PC54

Shipping and Storage

-20°C

Components

Component	PC54 500U	PC54 2500U	PC54 10000U
HotStar Best DNA Polymerase,5 U/μL	100μL	5×100μL	2×1mL
5×HotStar PCR Buffer	1.9mL	5×1.9mL	8×5mL

Note: The 5×HotStar PCR Buffer of this product contains 8.5 mM magnesium ions.

Description

This product is a chemically modified hot-start high-fidelity polymerase. The polymerase has 5'-3' DNA polymerase activity, 5'-3' exonuclease activity and 3'-5' exonuclease activity. Under normal PCR conditions, compared with Golden Star Taq DNA Polymerase, it has higher amplification efficiency excellent performance with high and low mismatch rate. The chemically modified enzyme has no polymerase activity at room temperature, which can effectively avoid non-specific amplification caused by the non-specific binding of primer and template or primer dimer under normal temperature conditions. The activation of the enzyme must be incubated at 95°C for 10 minutes his which can be integrated into existing PCR thermal cycling programs. The optimized buffer system maximizes the effect of the enzyme to achieve high fidelity, high specificity, high amplification efficiency, and high sensitivity amplification of the target fragment. Most of the PCR products amplified with this product have an "A" base attached to the 3' end, so they can be directly used for T/A cloning. This product is used in conventional PCR, RT-PCR and multiplex PCR,especially for PCR with high requirements on specificity, fidelity and amplification efficiency

Active Definition

Using activated salmon sperm DNA as template/primer, the amount of enzyme required to incorporate 10nmol of deoxynucleotides into acid insoluble substances within 30 minutes at 74°C was defined as 1 activity unit (U).

Quality Control

After several column purifications, the purity of the SDS-PAGE test is more than 99%; no exogenous nuclease activity is detected; no host residual DNA is detected by PCR method; it can effectively amplify single-copy genes in the human genome; it can be stored at room temperature for one month , no significant activity changes.

Protocol

The following example is a PCR reaction system and reaction conditions for amplifying a 1kb fragment using human genomic DNA as a template. In actual operation, corresponding improvement and optimization should be carried out according to the different template, primer structure and target fragment size.

1. PCR reaction system

Reagent	50μL reaction system	Final Concentration
5×HotStar PCR Buffer	10μL	1×
dNTP Mix,10 mM each	1μL	200μM each
Forward Primer,10 μM	2μL	0.4μM
Reverse Primer,10 μM	2μL	0.4μM
Template DNA	< 0.5μg	< 0.5μg/50μL
HotStar Best DNA Polymerase,5 U/μL	0.5μL	2.5 U/50μL
ddH2O	up to 50μL

Note:Please use the final concentration of 0.1-1.0μM as the reference for the setting range of primer concentration. When the amplification efficiency is not high, the concentration of primers can be increased; when non-specific reactions occur, the concentration of primers can be decreased to optimize the reaction system.

• PCR reaction condition

Step	Temperature	Time
predenaturation	95°C	3min
denaturation	94°C	30s
annealing	55-65°C	30s	30-40cycles
extend	72°C	60s
final extension	72°C	5min

Note:1) In general experiments, the annealing temperature is 5°C lower than the melting temperature Tm of the amplification primer. When the ideal amplification efficiency cannot be obtained, the annealing temperature should be appropriately reduced; when non-specific reactions occur, the annealing temperature should be increased to optimize the reaction Conditions.

2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of HotStar Best DNA Polymerase contained in this product is 1-2 kb/min.

3) The number of cycles can be set according to the downstream application of the amplified product. If the number of cycles is too small, the amount of amplification will be insufficient; if the number of cycles is too many, the probability of mismatching will increase and the non-specific background will be severe. Therefore, the number of cycles should be minimized on the premise of ensuring the product yield.

4) This product must be pre-denatured at 95°C for 10 min to activate the enzyme.