I've been doing colony PCR for last few days but haven't seen any bands on the gel. I've tried both positive and negative controls, but I'm still not getting any bands. Here's my procedure: I use a pipette tip to collect single colonies from the selection plate, restreak them on a fresh LB agar selection plate, and then swirl the remaining cells in 50 µL of sterile water. I then boil the mixture at 95°C for 30 minutes. In the PCR, I use 1 µL of the boiled cell-water mixture as the template.
for the thermal cycler, I started with 10 min at 94 degrees then I used 53 degrees for annealing temperature and a total of 35 cycles. I used gel load dye and added 5 micro to 25 micro samples and the ladder was NEB TriDye 1 kb+. 2 micro of the ladder was used. I don’t know what is the problem with my pcr. Are there any suggestions?
I agree with Elizabeth Pradel that you do not need to boil that long. I usually did 3-5 minutes only. However sometimes you have too much colony material and there are some inhibitors present. When my controls failed I would just dilute the template a bit more (try 5-fold dilution in water) and use 1 ul of that. Plasmid template is always in vast vast excess so it won't change your amplification.
Krishna Nandakumar your PCR protocol as you present it makes little sense :-(
I suggest:
Suspend a tiny amount of the colony in the water. The turbidity should be visible, but only hardly.
Vortex briefly and transfer 10-20 ul into a PCR tube
Add PCR mix (or buffer+nucleotides+enzyme)
PCR program:
4 minutes 95C
-----------
Loop 30 times:
30 seconds 95C
30 seconds annealing (53C?)
1 min per kb 72C
-------------
72C 5-10 min
Run.
Do a positive control on purified DNA, if this doesn't work check your conditions!
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