Standard PCR Master Mix 2X (with dye) contains all the components required for a successful polymerase chain reaction, which includes appropriate amounts of dH2O, DNA polymerase, dNTPs, MgCl2, and reaction buffers for effective amplification. This master mix formulation saves time and reduces contamination due to a reduced number of pipetting steps required for PCR set up. The mix is optimized for efficient and reproducible PCR, and After the PCR reaction is completed, all components are adequately tuned and ready to load on the gel (NO LOADING DYE NEEDED). The master mix contains polymerase which is a high-fidelity polymerase. In most cases, the error rate is 1 in every 1.3 million base pairs. At 72°C, the DNA Polymerase has a processivity of roughly 1 kb/min. It is compatible with TA cloning.
Standard PCR Reaction mix with 2X PCR Master Mix
Component
For 25ul reaction
For 50ul reaction
Final Concentration
Template (DNA)
variable
variable
<1,000 ng
10 μM Forward Primer
0.5 μl
1μl
0.2 μM (0.05–1 μM)
10 μM Reverse Primer
0.5 μl
1μl
0.2 μM (0.05–1 μM)
2X PCR Master Mix
12.5 μl
25 μl
μl 1X
Nuclease Free water
to 25 μl
to 50 μl
Standard PCR Condition
Step
Temperature
Time
Number of Cycles
Initial Denaturation
95°C
1–2 minutes
1 cycle
Denaturation
95°C
0.5–1 minute
25–35 cycles
Annealing*
42–65°C
30 seconds
25–35 cycles
Extension**
72–74°C
2–4 minutes
25–35 cycles
Final Extension
72–74°C
5 minutes
1 cycle
Soak
4°C
Indefinite
1 cycle
* The annealing temperature for a specific amplification reaction will depend upon the sequences of the two primers. See Section III for discussions on determining optimal annealing temperatures for PCR amplification. ** Allow approximately 2 minutes for every 1kb to be amplified.
Result:
Fig: Pant genotping using different Pcr master mix available. 2X PCR master mixed from Gene to protein Pvt Ltd (Lane 4 and 11) worked equally good as compared to brand A (Lane 2) and B (Lane 3) for gene 1 and could amplify the desired band efficiently.
