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?
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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