Does anyone have advice for performing PCR?

Question

I am trying to extract DNA from an insect - I am not getting any bands except for very very faint ones. Suggestions? Thanks!

Answer 1

First of all make sure your template is good quality. Did you extract genomic DNA, do you have a library in plasmids...(?).
Try re-purifying your template.

Maybe your template is GC-rich or has some secondary structure.
Is your template very long?If so make sure you let the polymerase is working at the elongating temperature for enough time (1min/kb)

If you are using plasmid template try not to use too much. Don't put more than 30-40ng DNA in a reaction.

Check your primers:
-can they form a dimer?
-can they anneal at more than one site on the template DNA with a only few mismatches?
-is the difference in the Tm of the two primers more than 6 C (it shouldn't;ideally they should be the same and I try not to exceed a difference of 4)?
-do your primers end in more than 3 c or g in a row?
-what's their Tm and GC content?Tm should not be very low (I design my primers at 56-61) and GC 40-60%.

The most strigent conditions are using as annealing temperature 2 C below the lowest Tm of the two primers. Try lower temperatures.

You can try different enzymes Taq, Pfu, Pwo, etc, even mixtures. Not all enzymes have the same processivity.

You can try different Mg+2 concentrations.

There are a lot of control experiments you can do depending on your template and primers. Eg if you have a library in a plasmid that has sites for universal primers you can use one such primer and the other respective primer you designed in order to check if there is something wrong with the quality of your primers.

If everything is perfectly designed and you've tried evreything but still get a very faint (but of correct size) band, gel purify it and use it as a template for a new PCR. In this case you'll have a very high chance of getting mutations so you'll need to use a high fidelity enzyme like Pwo or Pfu turbo and of course sequence a lot till you get a correct clone.

Answer 2

If you are getting banding after PCR your PCR worked so it is not so much your procedure. Try running a gradient with your primers to find an annealing temperature which gives you the expected product size. If you still have no luck, take another look at your primers, watch for primer-dimer, and make sure that if you designed the primer off of other species that it is degenerate or highly conserved enough to bind to your species of interest.

Answer 3

It's spelled CPR, dummy. And no, I've never performed CPR on an insect. I mean mouth-to-mouth on a bug? Oh, grody! And where would you do chest compressions? And how can you do them without crushing the whole bug. And then there's that nasty stuff all over your hand. Ewwww.

Answer 4

Watch your temperatures, GC content, and take adequate measures to avoide contamination.

Answer 5

no