PCR Amplification from Microbial Colonies

RAPD PCR Colony Miniprep

1. Edit the program or pick up a program (No 10) if the program has been set.
   Reaction time for RAPD PCR.
   1 cycle : Denature 95 C for 2 min
   45 cycle of
   Denature 94 C 15 sec
   Anneal 40 C 30 sec
   Extend 72 C 90 sec
   1 cycle of 72 C for 5 min
   4 C hold
   
2. Inoculate a touched-bacterial colony with a sterile toothpick into 100 µl of sterile H2O and voltex the tube.
3. Boil (95 C) for 5 min in the| water bath
4. Cool on the ice for 1-2 min and voltex.
5. Spin (14 K, 5 min).
6. Withdraw 2 µl of the sample (supernatent), add to 16 µl H2O in the 0.2 ml tube, and add a mixture of 2.5 µl 10X PCR buffer, 2 µl 2.5 µM primer , 2 µl 2.5 mM dNTP (2.5 mM each of dATP, dCTP, dGTP, and dTTP) and 0.5 U AmpliTaq.
7. Seal the tube cap.
8. RUN to start the program.
9. After the whole cycle, add 2.5 µl stopping dye.
10. Withdraw 10 or 12.5 µl of sample with a P-20 pipet and subject to electrophoresis (1.2 or 1.4 % agarose gel, 160 V) for 1 hr.