PCR for mCherry

Materials

1. Q5TM High-Fidelity 2X Master Mix (DNA polymerase, dNTPs, Mg++, buffer)
2. Forward primer
3. Reverse primer(2 kinds)
4. Template DNA (pBX-084 PB5 - HS4 - TRE - mCherrynuc - 2A - BlaloxN - GpA - HS4-PB3)
5. dd H2O

Methods

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. The total volume is 300 μl, including 150 μl Q5TM High-Fidelity 2X Master Mix (dilute it from 2X to 1X), 3μl forward primer, 3μl the first kind of reverse primer, 1 μl template DNA and 143μl dd H2O. And divide the 300 μl solution into 6 PCR tubes in average. Attention, the operation should be finished on ice.
4. The same to procedure 3, just change the first kind of reverse primer into the second kind of reverse primer.
5. Set the PCR machine and run:

   Stage1: Initial denaturation: 98°C，30s.

   Stage2: Denaturation :98°C，10s

   Anneal: 55°C，57°C，59°C，61°C，63°C，65°C，respectively.15s.

   Extension: 72°C, 30s.

   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.

Results

According to electrophoresis results, the optimum anneal temperatures for these primers are 55°C，57°C , while for 59°C and 61°C the quantity of product is relatively low. Under 63°C and 65°C, the number of product is less.