Should I clone my PCR product even though it is invisible on gel? - (Aug/09/2006 )
I've got 2 co-workers telling me 2 different things. Is it possible to have a product, but not be able to see it on a gel? This is just regular diagnostic PCR. One person is telling me to clone my PCR product even though I can't see it on the gel. Another person is telling that doing that would be a huge waste of time.
IMHO, I would guess that the second is correct. there is a chance that you have product...but your concentration will be very very low. so, your odds of getting the correct transformant after all that work are also pretty low. you might get it, but you might waste a lot less time if you just started over with more product.
If u have a good imager, u should b able to c if ur DNA conc. is as low as 2-3 ng. If its lower, then it would b difficult to use it for cloning.
I would agree with the second co-worker.
spike another pcr rxn with your first pcr rxn. If the first pcr just didn't ramp up enough product - the second one should. If you don't see anything the second time you can probably be assured that you saved time from having to ligate and screen a bunch of colonies that may or may not have the correct construct.
Cut your losses and start again. Remember, if you have done 30 cycles of PCR, you have amplified your original template over a billion-fold. If the product is not there, its probably not there... Doing another 30 cycles is likely to introduce more errors.
You should start to do some troubleshooting. Chuck out all of your reagents, make fresh dilutions and try again. If it still doesnt work, check your primers, and think about cleaning up your template.
Thanks for your help. I forgot to mention that these are formalin-fixed samples. I don't know if that makes a difference. All my reagents work fine with fresh samples so I don't think that's the problem. I'll try cleaning my template and possibly doing a second pcr rxn to see if that helps. I'm redoing the DNA extractions today and adding some extra washes to see if maybe I'm getting some inhibition from the formalin.