Hi everyone, not sure if this has been answered yet.
So I am trying to prepare a library for transposon mapping in a yeast using the nebnext ultra kit. I fragmented the DNA into what should be an average of 350bp in length. While I ran through the procedure I took 1ul out at after size selection after ligation and 1 ul after amplification for a BioA run. I got some strange results.
It seems like my size selection was a little off possibly due to my fragmentation not working the way I thought it did.
As you can see in both samples I had fragments + linkers from 200 to 500bp. indicating a fragment range around 50 to 350, which I may be able to further optimize.
The part that really confused me however is the post PCR BioA. In both samples I had what looked like very little amplification. however I see that I have two huge peaks in both samples one at ~126 and one at ~140. The PCR was run with 10 cycles as the starting material was quite low. I was wondering if anyone had any idea as to what either of these to peaks were. I thought at first they were adaptor dimers but any evidence of them are absent in my post ligation BioA.
Sample 3 and 5 are paired as post ligation and post PCR respectively.
4 and 6 are paired as post ligation and post PCR respectively.
samples 1 and 2 were unrelated to this problem and the other samples are not mine.
Any help would be much appreciated and I can clarify any questions about the procedure or samples if necessary,
Andrew
So I am trying to prepare a library for transposon mapping in a yeast using the nebnext ultra kit. I fragmented the DNA into what should be an average of 350bp in length. While I ran through the procedure I took 1ul out at after size selection after ligation and 1 ul after amplification for a BioA run. I got some strange results.
It seems like my size selection was a little off possibly due to my fragmentation not working the way I thought it did.
As you can see in both samples I had fragments + linkers from 200 to 500bp. indicating a fragment range around 50 to 350, which I may be able to further optimize.
The part that really confused me however is the post PCR BioA. In both samples I had what looked like very little amplification. however I see that I have two huge peaks in both samples one at ~126 and one at ~140. The PCR was run with 10 cycles as the starting material was quite low. I was wondering if anyone had any idea as to what either of these to peaks were. I thought at first they were adaptor dimers but any evidence of them are absent in my post ligation BioA.
Sample 3 and 5 are paired as post ligation and post PCR respectively.
4 and 6 are paired as post ligation and post PCR respectively.
samples 1 and 2 were unrelated to this problem and the other samples are not mine.
Any help would be much appreciated and I can clarify any questions about the procedure or samples if necessary,
Andrew
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