Enrichment problems

[h2jhwang]

Our customer submitted randomly amplified DNA. Customer said that the dsDNA was synthesized from RNA and DNA. The size of sample is from 100bp over 2.5 kb. I prepared a libray using rapid library preparation method. The libray size was bigger than a library which I prepared using genomic DNA. When I did emPCR but I can't get enough sequencing beads even at 64 cpb. I also try to do emPCR at 4, 12, 24 cpb. I can't get enough sequencing bead. When I did emPCR at 4cpb or 6cpb using a libray of genomic DNA, I got enough sequencing bead.

Do you have any idea about the libray prep. and emPCR? Or does anybody have sequencing experience using randomly amplified DNA?

Hong

[Aniki]

you should try to run the library on a bioanalyzer and see a definite shift by approximately 50-70 base pairs once you have finished your library preparations. It is possible that you have not properly added the adapters and that can easily explain why you are unable to achieve proper titration values.

[h2jhwang]

Thanks Aniki

I ran the library on bioanalyzer, first. The library was out of range (peak was over upper marker) of DNA sensitive chip and I asked roche support team. They recommened to run argarose gel. They said to go sequencing. But I did not get good result.

If adaptor was not add properly, why?

When I sequenced genomic DNA, I never meet this problem. Just this type of sample (ramdomly amplified cDNA frgments which were evenly distributed from 100 bp to 3 kb) has problem.

Do you have any idea what problem is?

[Aniki]

I have not really encountered this problem before. In theory it should be fine. I suggest you start from stock DNA and try again, this time to be sure to follow up with bioanalyzer runs showing that your DNA has indeed shfted ~70 bp upward. It isn't normal to use 24cpb and not have amplification, UNLESS you simply do not have the adapters. In that case you cannot amplify anything if they are not annealing.

Good luck. Perhaps call Roche once more?