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Help with Phenol-Chloroform DNA Extraction


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#1 morningface

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Posted 06 October 2009 - 02:34 PM

Hello,

I have been using a Phenol-Chloroform protocol to extract DNA from soil, and then precipitating with 10% NaAcc and EtOH. This time around, after adding the NaAcc and EtOH and allowing the DNA to precipitate at 4C overnight, I spun the samples down and found that there were two layers in 49 out of 50 samples. The top layer is the EtOH and is completely clear, there is a dark demarkation between the two layers, and then a tiny lower layer that is slightly yellow. I suspect that the lower layer is phenol, but I'm unsure as to how to proceed. Is there a way to get rid of the phenol? Where is my DNA? Help!

#2 DC Girl

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Posted 09 October 2009 - 07:18 AM

You can pipette the top aqueous layer into a new tube (this should contain your DNA). Be careful not to pipette any of the phenol. It's best to leave some of the top layer and have a lower yield than to contaminate the DNA with phenol which will affect downstream applications.

After pipetting off the top layer, you can prepitate it again with ETOH and NaAc, centrifuge and than resuspend your pellet in water or buffer.

I hope this helps.






Hello,

I have been using a Phenol-Chloroform protocol to extract DNA from soil, and then precipitating with 10% NaAcc and EtOH. This time around, after adding the NaAcc and EtOH and allowing the DNA to precipitate at 4C overnight, I spun the samples down and found that there were two layers in 49 out of 50 samples. The top layer is the EtOH and is completely clear, there is a dark demarkation between the two layers, and then a tiny lower layer that is slightly yellow. I suspect that the lower layer is phenol, but I'm unsure as to how to proceed. Is there a way to get rid of the phenol? Where is my DNA? Help!



#3 phage434

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Posted 09 October 2009 - 02:41 PM

Yes, avoiding carryover of the phenol is important. If you need high yield, you can take the supernatent, then add water to the remaining phenol, vortex, and take the supernatent again. I would also recommend at least one extraction with chloroform only following a phenol/chloroform extraction to remove phenol from your sample.

#4 Serkan

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Posted 10 October 2009 - 06:50 AM

as far as i understand you, your DNA sample have contaminated with phenol.
at your hand procedur, after phenol step carry out, you should go on next step again (extraction with chloroform).

because you know that chloroform hold phenol than when you spin tube, phenol+chloroform complex sample out
after new tube carefully pipetting the top layer, you can precipitate it again with NaAc and EtOH,centrifuge and
than resuspend your pellet in water or buffer (i advice "10 mM Tris")

i hope this help...




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