DNA quantitation & library preparation problem

[charlescoldroom]

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!

[luc]

Yes, I would seriously look for new samples (the heat denaturation of the Prot. K is not necessary when doing Phenol/Cholorform extractions - the phenol denatures the enzyme) . The truseq protocol (like all others I am aware of) requires double-stranded DNA. You could incubate your already denatured samples for four or more days in 0.05x SSC at 37C and hope that most single copy sequences renature - but this process will likely be rather imperfect.

[charlescoldroom]

Thanks for the answer luc!
I will try a couple of samples in SSC just to see what happens, but for the rest where I can I will prepare new DNA.
Thanks again!

[balaji_chattopadhyay]

Hi,
I am working with genomic DNA of mammals. I am trying to standardize RADTAG protocol. In this regard I need to know the importance of genomic DNA concentration for restriction digestion and the least quantity of genomic DNA that can be used. I use Qiagen DNA extraction kit and my samples have typically shown ~ 10ng/ul concentration in nanodrop. But flourimeter (Qubit), returned genomic DNA concentration of~2ng/ul. The radtag protocols available usually refers to starting quantity of 100ng to 1ug, with most reports suggesting 1ug. At the most I can try to concentrate my DNA and may obtain ~50 to 100 ng yield for a 50ul restriction digestion experiment.
It would help me much if I can get some advice regarding possibilities of standardization. My samples are obtained through non lethal sampling of wild caught animals and it may not be possible to obtain more tissue or repeat sampling.

[luc]

Hi, the nanodrop is extremely unreliable at DNA concentrations of about 20 ng/ul and under. The Qubit data are a lot more trustworthy.
If your isolated DNA is of reasonably good quality it might be possible to use whole genome amplification (WGA - there are several phi -polymerase based kits out there) to increase your sample. You can get very representative sample amplifications from 20 ng starting material.

[balaji_chattopadhyay]

Thank you very much luc for your reply. I do not know anything about phi- DNA polymerase kits. Do you think Phusion high Fidelity PCR master mix from New England Biolabs would be useful??
I also wanted to know if you suggested that i can start with as low as 20ng for restriction digestion.
thank you in advance

[luc]

These are the WGA kits I was writing about:

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-US/products/AlternativeProductStructure_17092/25660030

http://www.qiagen.com/products/REPLI-gMiniKit.aspx

WGA is very different from PCR for which the Phusion polymerase is used.

Wikipedia has a good intro to WGA:

http://en.wikipedia.org/wiki/Multiple_displacement_amplification

[pmiguel]

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...

Check my earlier post out.

It shows a nanodrop spectrum of 0.1% pure phenol dissolved in water. (I pipetted 1 ul of molten pure phenol into 1 ml of water). Nanodrop reads it as having a "DNA" concentration of > 0.5 ug/ul! 260/230 is well above 2.6, and 260/280 is 2.2 yet it contains no nucleic acids at all. The ratio you want to look at is 260/270. If this is below 1.2 or so, you probably have residual phenol in your sample. 0.1% is enough to completely confound attempt to determine the actual concentration of any nucleic acids in your samples. But even lower amounts will cause issues.

--
Phillip