PCR disappeared - there is no amplification (Nov/20/2008 )
Hello everyone... I have a huge problem... I'm sequencing kras gene but it have past almost 2 months since i have no amplification on my PCR... first i found a contamination on my negative control, I was trying to clean it up and I changed everything, then I begun to have an unespecific amplification on the botom of my PCR but every time this thing turned more intensive and my PCR product begun to disappear...
I'v changed everything again and again and I can't resolve my problem. First I was using Platinum Taq fon Invitrogen and now I'm trying with Gold Taq fom Applied Biosystems and with this one I obtain 2 amplification but whene I Purify this I lost everything because the rim is very low...
Please help me.....
I'v changed everything again and again and I can't resolve my problem. First I was using Platinum Taq fon Invitrogen and now I'm trying with Gold Taq fom Applied Biosystems and with this one I obtain 2 amplification but whene I Purify this I lost everything because the rim is very low...
Please help me.....
Changing things sometime works but u can try different annealing temp or also try with different concentration of primer,Mg etc... try to do gradient pcr//
It appears you've changed everything but the primers. Have you tried a new (re-designed) set of primers?
Are you using the same tube of DNA? Maybe its time to extract a new batch. Repeat freeze thawing might affect your template quality.
There seems to be something wrong with the primers.
-meredith
perhap you should adjust the annealing temp. try to make the temp ladder
I'v changed everything again and again and I can't resolve my problem. First I was using Platinum Taq fon Invitrogen and now I'm trying with Gold Taq fom Applied Biosystems and with this one I obtain 2 amplification but whene I Purify this I lost everything because the rim is very low...
Please help me.....
Changing things sometime works but u can try different annealing temp or also try with different concentration of primer,Mg etc... try to do gradient pcr//
I'v try with the annealing temperature also, first i was working with 58.8'C, i'v decrese the temp to 57.5 cuz 57 is too low, I lost everything... I'v increased Mg concentration from 2 to 4mM, now i have 2 bands...
did it too... new primers but not amplification
I agree with chrisbelle. It could be very likely that your DNA has degraded and is no longer suitable for PCR amplification- especially if you have been freeze thawing the template multiple times in two months.
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.