Hi All:
I'm preparing libraries for the HiSeq...
We're generating a library of variants, and the whole construct is 143nt long...with 30 nt in the middle that are variable (lots of variants!).
Trying to adapt the TruSeq protocols to my application, I've already had to resort to using Qiagen Minelute columns instead of AmpPure (my size is below the threshold (all in the wash)).
I am planning on producing 1ug of DNA (via PCR, unfortunately) to then do end-repair, phosphorylation, ligation, etc...
Am I on the right track here...or should I assume loss after shearing? By 'loss', I mean that not all gDNA is at the correct size after shearing, but I know my PCR'd DNA is All the right size...or do I just go forward with 1ug of PCR'd DNA?
Also, since my final size is going to be smaller than suggested, should I alter amplification/extension conditions in the cluster station? I'm not running the machine, so what should I tell the tech?
Thanks!
I'm preparing libraries for the HiSeq...
We're generating a library of variants, and the whole construct is 143nt long...with 30 nt in the middle that are variable (lots of variants!).
Trying to adapt the TruSeq protocols to my application, I've already had to resort to using Qiagen Minelute columns instead of AmpPure (my size is below the threshold (all in the wash)).
I am planning on producing 1ug of DNA (via PCR, unfortunately) to then do end-repair, phosphorylation, ligation, etc...
Am I on the right track here...or should I assume loss after shearing? By 'loss', I mean that not all gDNA is at the correct size after shearing, but I know my PCR'd DNA is All the right size...or do I just go forward with 1ug of PCR'd DNA?
Also, since my final size is going to be smaller than suggested, should I alter amplification/extension conditions in the cluster station? I'm not running the machine, so what should I tell the tech?
Thanks!
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