good amplification in classic PCR, no amplification in qPCR
Posted 19 March 2010 - 06:06 AM
I have troubles amplifying my samples. After a ChIp I purified the DNA on Macherey-Nagel NucleoSpin Extract II, eluted the samples with water and precipitated them with EtOH. I first realized a classic PCR using the Taq polymerase and I obtained correct bands, immediately after I started a qPCR using both MESA Green qPCR and AB Sybr Green PCR Master Mix. As an additional control I used control genomic DNA from a kit of ActiveMotif. The control gDNA was amplified but not my samples.
What can be the cause?
When I am measuring the absorbency of my samples before the PCR/qPCR, the 260/230 ratio is very low. Still the classic PCR with the Taq polymerase is working very well.
I also mixed my sample with the genomic DNA and had no amplification.
If you have any suggestions I would be very glad to know them
Thank you in advance!!!!
Posted 19 March 2010 - 08:13 AM
It's possible you have an inhibitor such as ethanol in your qPCR reaction - try diluting your template in water to see if this allows amplification.
Posted 19 March 2010 - 08:20 AM
I concentrated my samples to the max. and used 1ul in stead of 5 and it didn't help...
Posted 19 March 2010 - 09:16 AM
Posted 19 March 2010 - 09:26 AM
oh, the "gDNA" is a control human DNA from an amplification kit, it has nothing to do with my templates, I just wanted to be sure that the Master Mix is ok.
but are you using the same primers in your control than your template?
From the previous post, maybe your template concentration is too high, try different dilutions.
Posted 08 April 2010 - 07:14 AM
I have the same problem:
I run the traditional PCR and I have the expected bands: massively intense with a ridiculous amount of template in the master mix.
I run the qPCR with three times more of templates: absolutely nothing, or wierd curve....what's wrong?
Is it possible that the problem is in the way of how the machine "read"?
I mean I know that is impossible to quantify via spectrophotometer the ChIP samples, and the qPCR reading is similar way...That's might be mean that if I purified more my samples will be better? ( But my samples came from a MIni elute coulmn...)
Posted 08 April 2010 - 05:56 PM
So....trying diluting your samples 1:10 or so....let us know what happens!