Genotyping Master Mix datasheet

Genotyping 2X Master Mix

(Cat. No.: E008)

Description:

Genotyping Buffer and PCR system are specially designed for identifying target DNA fragment from complex genome background. It is about 10²-10⁴ times more sensitive than general PCR system, especially for high GC contents or DNA sequence with complex structure.

Genotyping 2X Master Mix is a premixed, ready-to-use solution containing Taq DNA Polymerase, dNTPs, MgCl₂ and Reaction Buffer at optimal concentrations for efficient amplification of DNA templates by PCR. The Taq 2X Master Mix has been optimized for use in routine PCR for amplifying DNA templates in the range of 0.2-4kb.

PCR Master Mix, 2X:

60 units/ml of Taq DNA Polymerase supplied in a proprietary reaction buffer (pH 8.5), 400µM each of dATP, dGTP, dCTP, dTTP, 13.4mM MgCl₂.

Features:

Fast:         Set up reactions in less than a minute.

Sensitive:     Amplify as little as 2 copies of target template.

Convenient:   One tube, one pipetting step.

Complete:       Reagents, including Taq DNA Polymerase, MgCl₂, dNTPs and buffers, in one tube.

Scalable:      Set up 10µl, 25µl or 50µl reactions.

Stable:         Stable for hundreds freeze-thaw cycles.

Performance Guarantee: SinoBio PCR systems, enzymes and reagents are proven in PCR to ensure reliable, high-performance results. If you are not completely satisfied with any SinoBio PCR product, we will send a replacement or refund your account.

Applications:

PCR. 3´ A-tailing of blunt ends, compatible with T-Vectors.

Unit Definition:

One unit of Taq DNA polymerase is defined as the amount of enzyme required to catalyze the incorporation of 10nmol of dNTP into acid-insoluble material in 30 minutes at 74^oC. The reaction conditions are: 50mM Tris-HCl (pH 9.0 at 25^oC), 50mM NaCl, 5mM MgCl₂, 200µM each of dATP, dCTP, dGTP, dTTP (a mix of unlabeled and [³H]dTTP), 10µg activated calf thymus DNA and 0.1mg/ml BSA in a final volume of 50µl.

Quality Control Tests:

PCR, activity, Bacterial DNA contamination, endo- and exo- nuclease, Specific performance test.

Storage:

Store at -20^oC.

Reaction Conditions:

Set up PCR reactions as follows:

Load thermocycler and run the following program:

The annealing temperature may be varied from 55^oC to 60^oC according to the Tm     £¦#118alue of your particular primers, and the extension time can be shorten to 1 min for short target fragment such as 500bp. But the thermofile list here is suitable for most cases.

Notes for application:

1.         User should be aware that the mutant ratio is higher than normal PCR system.

2.         Carefully design your target PCR fragments between 500bp-3000bp to get the optimized result.

3.         For the high sensitivity; sometimes there will be ghost bands appearing in your PCR. These do not seem to be significant as they have not been shown to be a problem.

Related Methods: Extract Genomic DNA for Genotyping:

1.       Cut about 10µg tissue or 10⁵ cell culture.

2.       Add 200µl Tissue Lysis Buffer, 55^oC over night.

3.       19,000 rpm centrifuged for 10 min.

4.       Transfer the supernatants into a new tube.

5.       Add equal volume of isopropanol. Mixed and centrifuged at 4^oC, 13,000 rpm 15min.

6.       Discards the supernatants and dried at 55^oC.

7.       Dissolved into 500µl distilled water at 55^oC for 5min. Use 1µl as template for PCR detection.

Tissue Lysis Buffer:

50mM Tris-HCl pH8.0, 5mM EDTA, 0.2% SDS, 200mM NaCl, 100µg/ml Protease K.