Hello!
I am kind of new in this NGS-business, struggling with library prep.
I prepared Testlibraries with ChIP'ed DNA using the TruSeq DNA Sample Prep Kit. I know that this kit is optimized for an DNA input of 1 µg, still I started with 10 ng.
Several changes I made to the original protocol including:
MinElute based purification
50-fold diluted Adapters
18-cycles of PCR
Furthermore I did the PCR prior to gel-based size selection.
From the gel I cut: 300-400 bp fractions (samples 4-6) and 200-300 bp fractions (samples 7-9 on bioanalyzer file) and purified the DNA with the MinElute Gel extraction Kit.
The library was validated using the High sensitivity DNA Kit on the Bioanalyzer.
I attached the bioanalyzer file. Please have a look at the samples 4-9.
Why are these peaks to spiky and why do I have another smear band higher than the desired fraction....
Advise is more than welcome...
Thanxx
I am kind of new in this NGS-business, struggling with library prep.
I prepared Testlibraries with ChIP'ed DNA using the TruSeq DNA Sample Prep Kit. I know that this kit is optimized for an DNA input of 1 µg, still I started with 10 ng.
Several changes I made to the original protocol including:
MinElute based purification
50-fold diluted Adapters
18-cycles of PCR
Furthermore I did the PCR prior to gel-based size selection.
From the gel I cut: 300-400 bp fractions (samples 4-6) and 200-300 bp fractions (samples 7-9 on bioanalyzer file) and purified the DNA with the MinElute Gel extraction Kit.
The library was validated using the High sensitivity DNA Kit on the Bioanalyzer.
I attached the bioanalyzer file. Please have a look at the samples 4-9.
Why are these peaks to spiky and why do I have another smear band higher than the desired fraction....
Advise is more than welcome...
Thanxx
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