PCR stopped working!!! HELP!!! - need results urgently (Jul/29/2009 )

i dont know what to do, i am so desperate!!! i have 2 months to turn in a bunch of results or else i wont get my maternity leave.

things were working just fine until one day, my pcr stopped working. ill brief my experiment:

part 1: using invitrogen one step do a first round to make cDNA and a first product with GSP (can not use random primers, they give many unspecific products in the final step)
part 2: using invitrogen platinum taq use the first as a template to make a final nested product with inner GSP

one fine day all the bands just disappeared! i have done the following:

- reextracted all my samples in case my template is messed up, this does not help
- changed all my reagents... zilch!
- tried using different primers as a positive control... and yes, they do work... under all the conditions and reagents ive tried.

so, how can my primers just "stop working" from one day to the next? i even tried having my primers made all over again and nothing! i dont understand.

can anybody please give me suggestions... as i said, i only have 2 months to finish a large bunch of samples, or else my boss will take away my maternity leave until i hand in results. "the reaction stopped working, ive tried everything" isnt an excuse..... please, please help!

Hi mjolner,

The fact that your control primers work is good. This tells you all your reagents, and samples, are in good working order.

It is not entirely uncommon for a set of primers to stop working all of a sudden. My recommendation is to check and re-check that you are using the correct primers. The ones you got re-synthesized may not be the correct ones. I am sure you are very organized and know for sure (100%) that the primers you re-ordered are the same ones that were working before. Yet, there is always a chance there is a mistake somewhere. My recommendation, based on your explanation, is to go back and confirm for the nth time that the primers you synthesized are truly the ones that used to work. Once you determine that they are the right sequence, order them from a different vendor (if you can). If this is not possible, order them again from the same vendor. It is not impossible for even the vendor to ship you primers that were not actually the ones you ordered (most vendors get tons of orders and switching orders in not impossible). I would even go as far (paranoid) as ordering a second set of primers you know work with your new order, that way you can check that the new synthesized primers come from a good batch.

Good luck

thanks for the uppers!

yes actually, i have already sent the primers to be remade and double checked the seqcuence. same ones that used to work, but brand new. neither the old ones nor the new ones will work now. other primers from the same company have always worked for us, i trust them....

any more ideas? anyone! thx

My comment can appear fullish, and you would have done this earlier also ...but I will suggest to re-check all your things, procedure, PCR machine, PCR program .....EVERTHING a to z once again..do it yourself and also get it done from your colleague/s .
If your reaction has worked earlier, it should work now also..If at all it doesn't, can cloning your first product and using the plasmid for the 2nd reaction would be the option???

the template might have become degraded from nuclease contamination. This is one of the most common causes for a PCR to suddenly stop working.

If there is sufficient material, check your DNA template on a gel for any signs of degradation. You will see a long smear of low molecular weight DNA if the sample is degraded.

If degraded, reextract the DNA from fresh material.

yeah, ive double triple cuadruple checked everything and tried all combinations possible....
what plasmid are you talking about? i dont use a plasmid for my cloning, what i do is nested pcr on rna samples......

um... have you tried using a different PCR machine? It might be in need of servicing,

Do you have a PCR reaction which you can run on your template DNA that would be certain to give you a positive reaction?

PCR is indeed a nasty business from time to time.

Try diluting or increasing the conc of your sample . and PCR sometimes to suffer from template batch variation. if you could find ur old sample , perhaps u wanna use that for the PCR.

In any case, PCR machine itself is quite a problem too. there's one time i have six similar tube of reaction ( aliquoted from a mastermix , 100% identical) but from the PCR result only one out of 6 works. could be temperature control problem. who knows.

If you carry wish to carry out PCR directly over earlier PCR product, this sometimes doesn't work. Instead if you clone your first PCR product in suitable vector and then carry out PCR over the recombinant plasmid, it gives better results. Can anybody comment on whether this would be true in case of mjolner?

thank you all for your suggestions... still havent been able to get things to work, so any more ideas are grateful, but it really helps knowing theres support out there... even if its strangers online...
most of the things i have already tried, but i will recheck.... and brainstorm more possible problems.... if u think of anything else PLEASE let me know!!!
if i get the damn thing to work ill let u all know how the hell i did it....

thx!