PCR Amplification from Microbial Colonies

RAPD PCR Colony Miniprep

2. 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
4. Inoculate a touched-bacterial colony with a sterile toothpick into 100 µl of sterile H2O and voltex the tube.
6. Boil (95 C) for 5 min in the| water bath
8. Cool on the ice for 1-2 min and voltex.
10. Spin (14 K, 5 min).
12. 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.
14. Seal the tube cap.
16. RUN to start the program.
18. After the whole cycle, add 2.5 µl stopping dye.
20. 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.

Reference: Zon, L. I., Dorfman, D. M., Orkin, S. H. 1989. The Polymerase Chain Reaction colony miniprep. Biotechnique 77: 696-698.

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