PCR disappeared - there is no amplification (Nov/20/2008 )
QUOTE (audrey-is-adjective @ Dec 2 2008, 08:06 AM)
Are you killing the Taq? Remember not to add it to a solution with a high concentration of salt. Like, don't add the salt first and then the Taq (salt kills Taq!) Add the Taq last of all, when the water and buffer and stuff are already in there and mixed together. Then mix again.
Anyone else in your lab? Bum some degradable stuff off them like dNTPs and Taq. Remake your primer from stock. How many freeze/thaw cycles have you put your dNTPs through?
Try to make a master mix using conditions and supplies that you KNOW work, with different primers or DNA or whatever. Basically get ONE PCR working. Note the conditions, supplies etc. even if they're not applicable to the PCR you want to work. Use that master mix as a positive control, and use it to troubleshoot what's happening with the important one.
Anyone else in your lab? Bum some degradable stuff off them like dNTPs and Taq. Remake your primer from stock. How many freeze/thaw cycles have you put your dNTPs through?
Try to make a master mix using conditions and supplies that you KNOW work, with different primers or DNA or whatever. Basically get ONE PCR working. Note the conditions, supplies etc. even if they're not applicable to the PCR you want to work. Use that master mix as a positive control, and use it to troubleshoot what's happening with the important one.
I know is very rare... I'v done allmost everything, I've try to amplify another gene (RET) with the same Taq, buffer dNTPs, Mg and DNA Templete and it came beautifuly, but with RAS is not coming... I still think that the problemmust be the primers but why the PCR was coming before???? why does not come with the new primers (new synthesis, new lot number...)
I'm getting tired... don't know what to do... -Gabyjk-
QUOTE (Gabyjk @ Nov 25 2008, 02:45 PM)
did it too... new primers but not amplification
Did you get new primers or *new* primers?
In other words, did you just re-order a new stock of the same primers you'd tried before, or did you design brand-new primers that anneal to a different location on your sequence and order them?
If two sets of different primers have failed, I'd suspect an issue with your template DNA. If you've only used on set of primers, I'd suspect the primers...
-HomeBrew-