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Enrichment PCR Failure
Hi all,
For the past few weeks we've been unable to successfully amplify our post-library prep samples. We use the Apollo324 automated system to do the library preps, then do the PCR enrichment and Ampure clean-up by hand. What we've seen in the past is decent yield coming off the Apollo and better yield following PCR (10 cycles for most samples) and clean-up (as one would expect!) But rather abruptly it seems that our PCR amplification has just stopped working. Instead of increased concentration following PCR, we have greatly reduced concentration, to the point where there's not enough sample to load on the HiSeq. I have a couple pictures of Bioanalyzer traces from the tests we've been doing this week, of the same sample before PCR amplification and then again after PCR amplification and clean-up, so you can see what I mean; we've seen samples worse than this as well.
One interesting aspect is that if we take the post-amplification sample and run another 10 cycles of PCR, we see concentrations more like what we would have expected to see after the first round of amplification (I don't have a bioanalyzer trace file for this, but we confirmed it with the nanodrop and an agarose gel). So apparently something is working with the ligation and the amplification - maybe just not very efficiently or consistently? Do you have any ideas on what could be causing the concentration drop after that first round of amplification?
(I did notice a similar thread from a couple years ago when I was searching the forums for ideas, but there was no resolution to the problem, so I figured it would be best to just start a new thread with my question. Hopefully that is ok!)
Thanks so much,
Kristina
For the past few weeks we've been unable to successfully amplify our post-library prep samples. We use the Apollo324 automated system to do the library preps, then do the PCR enrichment and Ampure clean-up by hand. What we've seen in the past is decent yield coming off the Apollo and better yield following PCR (10 cycles for most samples) and clean-up (as one would expect!) But rather abruptly it seems that our PCR amplification has just stopped working. Instead of increased concentration following PCR, we have greatly reduced concentration, to the point where there's not enough sample to load on the HiSeq. I have a couple pictures of Bioanalyzer traces from the tests we've been doing this week, of the same sample before PCR amplification and then again after PCR amplification and clean-up, so you can see what I mean; we've seen samples worse than this as well.
One interesting aspect is that if we take the post-amplification sample and run another 10 cycles of PCR, we see concentrations more like what we would have expected to see after the first round of amplification (I don't have a bioanalyzer trace file for this, but we confirmed it with the nanodrop and an agarose gel). So apparently something is working with the ligation and the amplification - maybe just not very efficiently or consistently? Do you have any ideas on what could be causing the concentration drop after that first round of amplification?
(I did notice a similar thread from a couple years ago when I was searching the forums for ideas, but there was no resolution to the problem, so I figured it would be best to just start a new thread with my question. Hopefully that is ok!)
Thanks so much,
Kristina
You seem to have experience in this area, would you please help me?
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