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Old 11-26-2012, 02:53 AM   #1
Papatya
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Location: England

Join Date: Aug 2012
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Question Problem with PCR amplification TruSeq DNA kit

Hi everyone,

I am new to Seq Answers but this seems like the right place to ask this question. I am currently preparing DNA libraries using the TruSeq DNA Preparation kit v2. I was getting very low yields (around 4nM) for the resulting library at the end and when I checked the concentration pre-enrichment-step I found that it was equal to the concentration after the PCR. So it seems the PCR is not working. The DNA that I am working with is very GC rich so I have made some alterations to the PCR (advised by others working with GC rich genomes). The alteration is only to the cycle:

1) ramp rate of 2.2 degrees Celsius/sec for all the steps
2) a denaturation time of 1 minute rather than 10s

Has any one else run into this problem? Is it likely to be that some reagents have gone off (the kit has been used twice before) or that the alterations I made are stopping amplification?
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Old 11-26-2012, 06:05 AM   #2
ETHANol
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Default

This is not your problem. It's something more serious but you should use the KAPA HF polymerase. It is way better with GC-rich templates then the TruSeq polymerase and way better in general.

As far as trouble shooting your problem you are going to have to provide a lot more detail.
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Old 11-26-2012, 08:03 AM   #3
kwaraska
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Default I agree

We have used Kapa HF master Mix for a couple of years and it has worked for everything. We are also currently testing the new NGS enzyme from NEB, but I do not have anything to report yet.
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