PCR HELP!!

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LearningGenetics
LearningGenetics's picture
PCR HELP!!

Hello, I am a new student. I have more of a ecology background but I am interested in plant population dynamics.

I just started conducting lab work, attempting to PCR my samples. The first batch that I did, did not work except for 3 lanes. I was wondering if any one had any idea where I should start. My first guess is that perhaps the concentrations of buffer and so forth might be off. I was just wondering if people have experienced this before and whether or not you had any laboratory suggestions.

Thanks

Omai
Omai's picture
Hello learning genetics,

Hello learning genetics,

A couple of questions:
Were the three lanes that work control lanes?
Was one of them the positive control for your primers?

It is critical that your positive control works. I wouldn't run any experimental lanes until I had my positive and negative controls working. Use a sample that you know contains your gene product. That way you can also confirm your digestion protocol and the concentrations for your polymerase, 10x buffer, primers, labeled nucleotides etc. . . The negative control can be just water instead of any sample. That way you will know if you are getting a false positive signal.

I've had luck increasing the amount of polymerase, and increasing the cycle number, if you don't see the expected band in the positive control lane.

Good luck and come back with any questions you have.

Omai