DNA extraction methods

[Bioinform]

hi i am a new member on this site. you guys are doing great job.
I will be doing a whole genome sequence of several grampositive bacteria and i need both the plasmid and gDNA on the isolate. I am using promega dna purification kit. Do you guys have any idea wheather this kit is able to isolate both the genomic and plasmid DNA. Or is there any other kit or methods with which i can isolate both of them.
Your help would be really appriciated.
Thank you

[ahmedmagds]

need for assistance

I need a kit for extraction DNA from gram-negative bacteria (Genome size is roughly 2.3 Mbp). The DNA will be used for next generation sequencing so The requirement for DNA samples are as below:
1) Sample condition: DNA samples without degradation and RNA contamination;
2) Sample quantity: ≥6ug
3) Sample concentration: ≥30 ng/µl
4) Sample purity: A260/280=1.8~2.0

[Jon_Keats]

Hi,

For library prep we use the Qubit values exclusively as long as it passed all the QC checks. I went through our database of results and pulled out the samples tested by both Nanodrop and Qubit. We see the following correlation (n=43) for percent quantity (Qubit/Nanodrop):

Median = 68%
Min = 38%
Max = 84%
STD = 8%
25th Percentile = 63%
75th Percentile = 70%
R-square = 87.4%

If you are only seeing 3-7% of nanodrop I'd want to ensure you have an RNA free prep and that it is not degraded. We have seen that degraded (smear on a gel) shows much lower concentration by qubit/picogreen than high molecular weight DNA at the same concentration as measured by nanodrop.

[gruberjd]

Hi all (especially Jon Keats)-- Thanks for all the participation in the thread. I also love the Qiagen Gentra Puregene kit for DNA extractions, but it seems that the dsDNA content (from Qubit) is lower than a more baroque non-kit approach (baroque about 2x as good dsDNA fraction, in my hands). BUT, I get far, far more DNA overall from Puregene, so I could more than compensate for this deficit. Puregene is also a lot faster and easier.

Does anyone have experience whether it is OK to simply trust the [Qubit] ug of dsDNA and ignore the fact that its only 3-7% of the [nanodrop] total DNA? If so, Puregene is the clear winner. I would still look to nanodrop to check the quality.

Thank you!

[LaKarN]

Hi !

I work on plants, we are about to start a RADSeq experiment, but suddently I have a question.

Do you think that cpDNA (chloroplastic) could "damage" the results, I mean too much representation of cpDNA versus nuclear DNA ?? Cause of course we need good depth coverage...

Did you hear something about how to limit organellar DNA extraction ?

Best regards

[Jon_Keats]

We commonly use a variety of DNA sources. Classic proteinase K followed by phenol-chloroform extraction and ethanol precipitation. Qiagen Gentra Puregene (my favorite method). Qiagen DNeasy (manual or from Qiacube). Invitrogen Purelink.

Like you mentioned QC is the most important issue. Nanodrop to get the basic quality values. We expect 260/280 1.8-1.9 for RNase treated DNA (always RNase treat) and want 260/230 >1.9. Then use Qubit to get the concentration (best to dilute to ~250ng/ul) by nanodrop so sampling is accurate. Then run ~200ng on a 0.6-0.8% gel to check the size of the DNA. You should have a nice clean single band of high molecular weight DNA above 20kb and no DNA stuck in the well.

[asis2]

Thanks both for your advice. I think they must have been referring to the automatic extractors as the DNA we are planning to use has been extracted from a clinical lab by an number of methods which we are trying to trace back. I guess QC steps will be important and will also purify with Qiagen.

Much appreciated!

[AJERYC]

I agree, Quiagen Kit (Maxikit) works fine for exome or genome sequencing. I heard from experts that automatic DNA extractors are not good for NGS since if you use them you need to further purify the DNA, for what you will use a Quiagen kit.

[adaptivegenome]

I use Qiagen Kits and have not had a problem. Whatever you use, just try to minimize fragmenting the DNA.

[asis2]

Hello,

I have heard that the method used to extract DNA originally can affect the final results on next gen sequencing, but I cant seem to find much information on this, can anyone advise me?

In particular I want to work with the illumina platforms hiseq/GAII on exomes through illumina enrichment, but am also interested in this problem more generally.

Thanks!

Ariane