Team:SUSTC-Shenzhen/Notebook/CRISPR/Point-mutation-PCR-for-UAS

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Team SUSTC-Shenzhen

Notebook

Elements of the endeavor.

Point-mutation PCR for UAS

2014/8/20


Materials

     Q5TM High-Fidelity 2X Master Mix (DNA polymerase, dNTPs, Mg++, buffer)
Forward primer
Reverse primer
Template DNA (PB5-PolyA-5xUAS-mcherry-lin-oct4-Neo--GpA-GpA-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

{{{2}}}

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

Verification by enzyme digestion

EcoRI digestion system

Component Volume
EcoRI 0.5ul
DNA 8.5ul
10X NEBuffer 2.1 1ul
Total 10ul

XbaI digestion system

Component Volume
XbaI 0.5ul
DNA 8.5ul
10X NEBuffer 2.1 1ul
Total 10ul

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

Gel electrophoresis

Making the agarose gel (2%)

Component Volume
TAE 40ml
Agarose 0.8g
Gene Green 1ul

Running conditions: 130V, 10min

Results of electrophoresis

{{{2}}}

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.

Maintained by the iGEM team SUSTC-Shenzhen.

Licensed under CC BY 4.0.