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Old 02-08-2017, 03:14 AM   #1
SeqTroubles
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Default CTAB DNA extraction - more RNA than DNA

Hi everyone,

Looking to get additional perspective on an issue driving me crazy. I am currently troubleshooting some library prep issues and am doing multiple extractions of one sample.

1 kit - MoBio ultraclean microbial DNA
1 kit - Qiagen DNeasy blood and tissue
1 CTAB + phenol chloroform

After the extractions I QC with nanodrop and qubit HS.

MoBio the results correlate nicely between the nanodrop and qubit. For the Qiagen and CTAB I get a tenfold difference in concentration.

I have done a gel and it appears to be the presence of RNA. I have already incorporated RNase A into the protocols. I double checked the efficacy of our stocks and they are working fine.
For the CTAB method the only thing I can think of is it might be contamination in the chloroform or the ethanol?

Thanks
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Old 02-08-2017, 05:49 AM   #2
ATϟGC
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What type of samples are you extracting from? I ask this because your poor correlations could also be due to co-precipitants like polysaccharides that give you poor A260/230 values. Ideally, A260/230 for DNA should be between about 2.0-2.2. I find that the more my DNA extracts are outside this range, the poorer correlations I get between nanodrop and qubit. CTAB-chloroform sometimes gives me very poor A260-230 values from polysaccharide contamination.
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Old 02-08-2017, 06:47 AM   #3
Olaf Blue
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Are you using Polyvinyl pyrollidone in any of the cleanup steps? Which plant/sample type are you extracting?
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Old 02-08-2017, 07:01 AM   #4
SeqTroubles
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Hi Thanks for the replys. Im extracting DNA from a gram negative bacteria species - Cronobacter. The absorbance readings for all extraction done do far are within good QC ratios. Some of the A260/230 are >2.2.
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Old 02-08-2017, 11:03 AM   #5
Olaf Blue
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OK. PVP shouldn't be necessary when extracting DNA from bacteria.
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Old 02-08-2017, 11:37 AM   #6
ATϟGC
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Hi again,

I usually see poor nanodrop-Qubit correlations when the A260/230 get below ~1.7. Here is a link to another thread discussing common contaminants and their effects on the spectra of samples in case it helps you determine what the cause is:

http://seqanswers.com/forums/showthread.php?t=21280
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Old 02-08-2017, 02:45 PM   #7
nucacidhunter
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NanoDrop quantification is based on absorption at 260 nm. RNA, dsDNA, ssDNA and nucleotides all absorb this wavelength. Qubit quantifies only dsDNA (using dsDNA reagent) so the discrepancy is due to presence of other nucleic acids, nucleotides and some contaminants.

If you add RNase with the buffer used for Resuspension then all the nucleotides from RNA degradation will be present resulting in higher read. The same applies to protocols that use RNase in a step that nucleotides are not removed in consequent steps. You can check following for effect of contaminates on NanoDrop readings:

http://www.nanodrop.com/Library/T009...ity-Ratios.pdf
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Old 02-09-2017, 05:15 AM   #8
SeqTroubles
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So I did another extraction yesterday with fresh chloroform and its the same problem. I used a pH strip to check out my P/C mix and it looks to be a definite pH of 7 maybe even slightly lower. So Im going to order in fresh P/C with a tris buffer and see how it goes.
Fingers crossed.
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Old 02-10-2017, 06:36 AM   #9
huguesparri
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Maybe I missed something but you said that you have "library prep issues".
Could you tell us what kind of protocol did you use to build your libraries?
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