DNA Extraction using Phenol and Ethanol - ETHANOL AND PCR (Dec/09/2008 )

Hi,

Desperatley need help!!!!!!!

I have extracted DNA from dirty water samples using phenol chloroform isoamyl technique, this involves using 70% ethanol to clear up my DNA at the end.

IMPORTANT QUESTION!!!

IF THE ETHANOL HAD NOT ALL BEEN DRIED OFF BEFORE I ADDED TE BUFFER WOULD THIS EFFECT MY PCR??

ALSO, IF THIS IS THE CASE - IS THERE ANYTHING I CAN DO TO GET RID OF THE ETHANOL SO THAT IT DOESNT CAUSE A PROBLEM WITH MY PCR?

THANK YOU

Janette

Hi,

Desperatley need help!!!!!!!

I have extracted DNA from dirty water samples using phenol chloroform isoamyl technique, this involves using 70% ethanol to clear up my DNA at the end.

IMPORTANT QUESTION!!!

IF THE ETHANOL HAD NOT ALL BEEN DRIED OFF BEFORE I ADDED TE BUFFER WOULD THIS EFFECT MY PCR??

ALSO, IF THIS IS THE CASE - IS THERE ANYTHING I CAN DO TO GET RID OF THE ETHANOL SO THAT IT DOESNT CAUSE A PROBLEM WITH MY PCR?

THANK YOU

Janette



Hey,
ethanol traces could interfere with all types of enzymatic reactions. I would recommend you to re-extract your DNA, if the pellet is visible, you can first pipette the ethanol and then either air dry at romm temperature for 20-30 min or use a speed vac although this is not recommended for genomic DNA as it would be difficult to redissolve it. I highly recommend Qiagen bench guide for tips and protocols

Hi Marzoq,

Thank you for your reply!

Firstly, I cannot re-extract the DNA.

My supervisor told me it was alright to add the TE buffer into my DNA even if there was some ethanol still present, but I think it is causing problems with my PCR?

Janette



Hey,
ethanol traces could interfere with all types of enzymatic reactions. I would recommend you to re-extract your DNA, if the pellet is visible, you can first pipette the ethanol and then either air dry at romm temperature for 20-30 min or use a speed vac although this is not recommended for genomic DNA as it would be difficult to redissolve it. I highly recommend Qiagen bench guide for tips and protocols

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The residual ethanol definitely could interfere with your PCR rxn. You just need to let it air dry for longer before you add your TE. I've heard that you can dry it at a higher than room temperature in a heat block but I've never tried this. Good luck.

Hi,

Desperatley need help!!!!!!!

I have extracted DNA from dirty water samples using phenol chloroform isoamyl technique, this involves using 70% ethanol to clear up my DNA at the end.

IMPORTANT QUESTION!!!

IF THE ETHANOL HAD NOT ALL BEEN DRIED OFF BEFORE I ADDED TE BUFFER WOULD THIS EFFECT MY PCR??

ALSO, IF THIS IS THE CASE - IS THERE ANYTHING I CAN DO TO GET RID OF THE ETHANOL SO THAT IT DOESNT CAUSE A PROBLEM WITH MY PCR?

THANK YOU

Janette

You could re-precipitate the DNA and work from there if all else fails.

Yes, you should be able to. Follow a protocol like EtOH DNA precipitation. Just remember to add a salt (Na acetate). Example protocol: http://userpages.umbc.edu/~jwolf/m5.htm