Store at -20°C,Avoid repeated freezing and thawing
Shipped In
Ice chest + Ice pads
Grade
EnzymoPure™
Product Description
MTaq DNA Polymerase is a novel DNA polymerase obtained by deletion of a segment of amino acid at the N-terminus of Taq DNA Polymerase and mutation modification. Through modification, this product can produce tolerance to inhibitors present in whole blood, allowing for direct amplification of DNA in human and mouse whole blood samples without the need for prior genome extraction and purification. The 3 'end of the PCR product is A and can be directly used for T/A cloning.
M665697
Component
500 U
2500 U
Storage
M665697A
mTaq DNA Polymerase, 5 U/μL
100 μL
5×100 μL
-20℃.Avoid freeze/thaw cycle.
M665697B
mTaq PCR Buffer, 10×
1.8 mL
5×1.8 mL
-20℃.Avoid freeze/thaw cycle.
Notice: mTaq PCR Buffer contains 30 mM MgCl₂
Quality control: After multiple column purifications, SDS-PAGE detected a purity of over 99%; No exogenous nuclease activity detected; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes in the human genome; Store at room temperature for one week without significant changes in activity. Usage: 1. Before use, please invert the mTaq DNA Polymerase repeatedly until completely mixed. 2. Place the PCR thin-walled tube on ice and add the following reagents except for whole blood.
Reagent
50 μlReaction system
Final concentration
mTaq DNA Polymerase
1 μl
/
mTaq PCR Buffer, 10×
5 μl
1×
dNTP Mix，2.5 mM each
4 μl
200 μM each
Forward Primer (10 µM)
2 μl
0.4 μM
Reverse Primer (10 µM)
2 μl
0.4 μM
Whole blood*
≤10%
/
RNase-Free water
x μl
/
Total
50 μl
/
Attention:
1) Before adding whole blood, repeatedly aspirate and beat up and down to completely mix various reagents 2) DNA template: Whole blood can be treated with sodium heparin, Na EDTA, K-EDTA, or sodium citrate. It is usually recommended to have a whole blood content of 5-10%. It is not recommended to use high concentration blood. For templates with high GC content, add 10% DMSO. 3) Primers: Oligonucleotide primers typically contain 20-30 nucleotides in length, with a GC content of 40-60% and evenly distributed within the primers. In conventional PCR reactions, the primer concentration should be at the final concentration of 0.1-1.0 μ M serves as a reference for setting the range. 3. Finally, add whole blood to the bottom of the tube. 4. PCR reaction conditions
Step
Temperature
Time
/
Pre denaturation
95℃
5 min
/
Denaturation
95℃
30 s
35-40 cycles
Anneal
50-68℃
30 s
35-40 cycles
Extend
72℃
250-500 bp/min
35-40 cycles
Finally extended
72℃
10 min
/
Attention: 1) Preheat the PCR instrument at 94-95 ℃ and place the sample on the PCR instrument to start cycling. 2) MTaq enhances cold sensitivity and has some hot start characteristics. Usually, the production of non-specific products can be avoided by preparing reaction ingredients on ice, adding polymerase, and preheating the thermal cycler to denaturation temperature (95 ℃) for immediate reaction. 3) Denaturation temperature and time: In order to fully lyse blood cells and release/denature DNA before the PCR cycle, an initial denaturation temperature of 95 ℃ for 5 minutes is required. 4) Annealing temperature and time: The annealing time is usually 30 seconds to 1 minute. The annealing temperature can be lower than the theoretical annealing temperature (Tm) by starting at 5 ℃ and optimizing through gradient PCR. 5) Extension time: The extension reaction is usually carried out at 72 ℃. Generally, the extension time is 1 minute every 250-500 bp. Recommend final extension at 72 ℃ for 10 minutes. 6) Typically, 35-40 cycles can achieve optimal amplification. 5. Result detection: After the reaction is completed, take 5 µ l of the reaction product and add it to the electrophoresis buffer for electrophoresis detection.
