qPCR problem

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kempf
kempf's picture
qPCR problem

Hi,

I've been running qPCR with Quantitect primers from QIAGEN. I've tried these primers a couple of weeks ago, and they worked great. However, I tried them again today (new cDNA, other reagents are the same as previous experiment), and basically everything amplified around cycle 30 (whereas before, I got amplification between cycles 19-25, depending on which gene I'm looking at). My triplicates are also terrible (difference in deltaCt up to 2 of 3 cycles sometimes), whereas they used to be quite good.
I think it's possibly a problem with my new cDNA, or it might be primer degradation (I resuspended them in sterile water instead of TE by mistake). Anyone got similar problems before?

Many thanks,

B

Ivan Delgado
Ivan Delgado's picture
 

 
Hi kempf,

What you describe can easily be attributed to degraded primers. Primers resuspended in water are much more unstable, and if you are storing them at 4oC they are likely at least partially degraded.

If you have some of your old cDNA around use it to see if you can get Cts of 19-25 again. If you do, then it is definitely the primers. Typically a new batch of cDNA can lead to higher Cts (>30), but not high Ct differences between replicates.

Good luck

Strosa8
Strosa8's picture
I have kept my cDNA samples

I have kept my cDNA samples in h20 for many months in -20C freezer, and I've never had a problem with degradation. cDNA is pretty stable, so most likely it isn't a problem with your template stability.  it could, however, be a problem with your cDNA quality.  if your 260/230 values aren't normal, any contamination can effect the efficiency of amplification of your primers.

vanshita
vanshita's picture
you are dissolving ur primer

you are dissolving ur primer in sterile water this is absolutly correct. but there maybe problm related to storage. i use to store my primer at -20. and cDNA is stable at -20. i think ur problm is because of primer degradation

fallah2010
fallah2010's picture
how do i make electro

how do i make electro-competent cell preparation

fallah2010
fallah2010's picture
how do I make heat- shock

how do I make heat- shock comtent cell preparation?

Ivan Delgado
Ivan Delgado's picture
 

 
For electro-competent cells, look at this protocol: 

http://ivaan.com/protocols/148.html

For heat-shock cells, look at this protocol: 

http://ivaan.com/protocols/135.html

fallah2010
fallah2010's picture
hello

hello
how do i make gel for electroforse preparative PCR?
thank you

Ivan Delgado
Ivan Delgado's picture
fallah2010, 

fallah2010, 

Please explain in more detail what you are trying to do. 

Cheers