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alcohol precipitation

alcohol precipitation - DNA Extraction Forum

alcohol precipitation - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


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Old 02-12-2014, 09:05 PM
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Question alcohol precipitation



Hello!

I'm working with a bead-beating method to get DNA from biofilm growing on Kaldnes K1 rings. I'm starting with 5 rings in extraction buffer (100 mM Tris, 100 mM EDTA, 100 mM phosphate, 1.5 M NaCl) and SDS in a bead-beating step. After bead-beating I've collected the supernatent and proceed with MoBio Powerlyzer DNA extraction kit at step 8. When I complete the DNA extraction, I can't see any DNA on a 1% gel nor quantify it with a nanodrop.

I contacted MoBio, and they had several recommendations. One of which is to alcohol precipitate the DNA to concentrate it after bead beating but before proceeding with the DNA extraction. I've found methods for ethanol and isopropanol precipitation of the DNA which typically recommend 0.3-0.5 M NaCl. Would the extra salt in my extraction buffer interfere with precipitation? Would diluting it with sterile DI water resolve the issue? Or would that complicate the issue by diluting the DNA as well? Do you have recommendations on which alcohol to use in the precipitation?

Thanks!
Eric
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Old 02-13-2014, 04:14 AM
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Default Re: alcohol precipitation

Because you are using the MoBio product, it is best that you ask them to provide you with specific protocol. As we may not know all the chemical they use. Importantly, will anything you do interfere with downstream process.

Generally, if you don't see any on gel or nanodrop i would suspect you don't have DNA at all. Or are you expecting a low yield? Did they suggest the amount for starting material?

Do update us if you have any new info. Thanks
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alcohol , alcohol precipitation , dna concentration , dna extraction , isopropanol , precipitation


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