Hello,
I am completely new to (deep) sequencing.
The project I am starting involves deep sequencing of SeqCap-enriched sequencing using the HiSeq sequencer.
I have a question...
To extract genomic DNA from tissue (my starting material) I have used a protocol which includes one step at 95 degrees for 10 minutes to inactivate the protease K, which is followed by classical Phenol/Cholorform purification and ethanol precipitation. The DNA is clean (>1.8 A260/280, >2.2 A230/260) as judged by Nanodrop. However, quantitation with Nanodrop is ~100 times higher than quantitation using the Qubit dsDNA HS. It has been suggested that the previous 95 degrees incubation could have completely denatured my gDNA...
Do you think that the isolated gDNA is not useable for library preparation using the TruSeq kit? Any suggestions welcome!
Thanks!
I am completely new to (deep) sequencing.
The project I am starting involves deep sequencing of SeqCap-enriched sequencing using the HiSeq sequencer.
I have a question...
To extract genomic DNA from tissue (my starting material) I have used a protocol which includes one step at 95 degrees for 10 minutes to inactivate the protease K, which is followed by classical Phenol/Cholorform purification and ethanol precipitation. The DNA is clean (>1.8 A260/280, >2.2 A230/260) as judged by Nanodrop. However, quantitation with Nanodrop is ~100 times higher than quantitation using the Qubit dsDNA HS. It has been suggested that the previous 95 degrees incubation could have completely denatured my gDNA...
Do you think that the isolated gDNA is not useable for library preparation using the TruSeq kit? Any suggestions welcome!
Thanks!
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