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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), 400M 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 10l, 25l or 50l 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 74oC. The reaction conditions are: 50mM Tris-HCl (pH 9.0 at 25oC), 50mM NaCl, 5mM MgCl2, 200M each of dATP, dCTP, dGTP, dTTP (a mix of unlabeled and [3H]dTTP), 10g activated calf thymus DNA and 0.1mg/ml BSA in a final volume of 50l.

Quality Control Tests:

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

Storage:

Store at -20oC.

Reaction Conditions:

Set up PCR reactions as follows:

Genotyping 2X Master Mix

25l

10M Primers

6l each

100-1000g genomic DNA

~1l

ddH2O.

to 50l

Load thermocycler and run the following program:

Step

Temperature

Time

Cycle

Heat Soak

93oC

90 sec

1

Denaturation

93oC

30 sec

40

Annealing

57oC

30 sec

Extension

65oC

3 min

Final

65oC

10 min

1

The annealing temperature may be varied from 55oC to 60oC 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 10g tissue or 105 cell culture.

2.       Add 200l Tissue Lysis Buffer, 55oC 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 4oC, 13,000 rpm 15min.

6.       Discards the supernatants and dried at 55oC.

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

Tissue Lysis Buffer:

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

 

 

 

For more information :: Bioxys and Gentaur BVBA :: Av. de l'Arme 68 B4 :: BE-1040 BELGIUM

Email: info@gentaur.com
 

International 

+32 (0) 16 58 90 45

+32 (0) 16 50 90 45

France

01 43 25 01 50

01 43 25 01 60

Italy

02 36 00 65 93

02 36 00 65 94

Germany

0241 6085 13140

0241 6085 33033

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Copyright 2005 Gentaur BVBA
Last modified: feb-07