PCR stopped working!!! HELP!!! - need results urgently (Jul/29/2009 )
mjolner on Aug 4 2009, 05:08 AM said:
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....
Hi, try to check your PCR temperature on the thermocycler whether is it accurate. Double confirm whether any jokers had messed up to your thermocycles internal settings like ramp rate etc. (something like this happened to me before)
Also, have you changed your PCR tubes to other brand lately? It might have a slight effect on your temperature. Some low quality brand had bad heat conduction activity.
Worst come to worst: try use miliQ water or nuclease free water in your PCR.
Also, you are using RNA as starting material...could it be your culture mutated, wrong expression condition etc?
just my 2 cents.
hi there... i got a hold of positive control primers for another zone that were able to verify that my templates are working.
i changed to a different taq buffer and now i get smudges with my primers that USED to work until a few weeks ago (and the new ones i sent to be made in case they had "died"), but nice bands with the positive control primers.
what could this be?
let me rephrase your condition
you are using RNA as starting material, convert to cDNA, done PCR. Used to be working.
1)now, same cDNA (from above), perform PCR: but not working; possible reason cDNA degraded. Your control is working probably your control is target a repetitive gene or some highly expressed gene in your genome.
2)if, not same cDNA as above, and you had re-extract fresh RNA from new sample, convert to cDNA: and done PCR but not working; check your experimental condition as your cells might not being induced to express the gene you want.
just some ideas that came across...