Point-mutation PCR for UAS

Materials

     Q5TM High-Fidelity 2X Master Mix (DNA polymerase, dNTPs, Mg++, buffer)

     Forward primer

     Reverse primer

     Template DNA (pBX-084 PB5 - HS4 - TRE - mCherrynuc - 2A - BlaloxN - GpA - HS4-PB3)
      
     dd H2O

Methods

Point-mutation PCR for UAS

1. Centrifuge the primers 12000 rmp for 5 min.
2. Dissolve the primers into dd H2O to the concentration of 50p mol/μl. Subpackage the solution for about 50 μl per tube to avoid repeated freezing and thawing.
3.Add 1μl template DNA into 9μl dd H2O to dilute the template DNA.
4. The total volume is 250μl, including 125 μl Q5TM High-Fidelity 2X Master Mix (dilute it from 2X to 1X), 2.5μl forward primer, 2.5μl the first kind of reverse primer, 1 μl diluted template DNA and 119μl dd H2O. And divide the 250 μl solution into 5 PCR tubes in average. Attention, the operation should be finished on ice.
5. Set the PCR machine and run:
Stage1: Initial denaturation: 98°C，30s.
Stage2: Denaturation :98°C，10s

     Anneal: 57°C，59°C，61°C，63°C，65°C，respectively.15s.

     Extension: 72°C, 10s.

     30 cycles.

Stage3: Final extension：72°C, 5 min.

     Hold: 4°C.

6. Check the product by gel eletrophoresis.
7. Keep the product in -20°C.

Electrophoresis for the product of PCR

According to gel eletrophoresis, the PCR is successful under all anneal temperatures.

Verification by enzyme digestion

EcoRI digestion system

XbaI digestion system

Incuabte at 37C for 4 hours [8.20 16:00pm~24:00pm]

Gel electrophoresis

Making the agarose gel (2%)

Running conditions: 130V, 10min

Results of electrophoresis

From the picture, we can see that, the position of digested PCR fragment aligned with that of the fragment cut down from the original plasmid, which indicated the success of our point-mutation PCR.