Ooops - chlorophom question- hope you can help me!?

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
User avatar
Ammie
Garter
Garter
Posts: 44
Joined: Wed Aug 16, 2006 6:48 am

Ooops - chlorophom question- hope you can help me!?

Post by Ammie » Mon Apr 16, 2007 4:36 pm

cDNA purification:

First step I was supposed to use a phenol/chloroform/isoamylalcohol solution. Mix and centrifugate and DNA is in the aquaeous phase.

Next step add chloroform. Mix and centrifugate and remove the DNA again.

I accidentally added chloroform in the first step, and then I thought that can a little bit extra chloroform hurt... it was in the mix anyway... så I continued and added the phenol-solution also and followed the protocol.

Is my cDNA ruined now or what do you think?

blcr11
Viper
Viper
Posts: 672
Joined: Fri Mar 30, 2007 4:23 am

Post by blcr11 » Mon Apr 16, 2007 6:45 pm

You haven't ruined your prep, but you may have residual phenol now. The phenol extraction removes protein. When you transfer the aqueous (upper) phase to a fresh tube, you are leaving any protein behind either dissolved in the phenol, or precipitated at the phenol/water boundary. Phenol is partially soluble in water, so you usually extract the aqueous phase from the phenol extraction once or twice with chloroform or chloroform:isoamyl alcohol (24:1) to remove any residual phenol. I would suggest that you re-extract your aqueous phase with chloroform or chloroform:isoamyl alcohol.

If all you did was to toss in the phenol on top of the chloroform and then proceded with the protocol, and so have already done an extraction with chloroform alone, then you're fine. Just precipate the DNA. If you haven't done a chloroform-alone extraction after any phenol extraction, then you may have residual phenol in you aqueous (DNA) phase--that's all I'm trying to tell you.

User avatar
Ammie
Garter
Garter
Posts: 44
Joined: Wed Aug 16, 2006 6:48 am

Post by Ammie » Tue Apr 17, 2007 11:11 am

Thanks!

I got a 260/280 quote of only 1,62. Could this be due to my misstake?

It is still worth testing in PCR isn't it?

blcr11
Viper
Viper
Posts: 672
Joined: Fri Mar 30, 2007 4:23 am

Post by blcr11 » Tue Apr 17, 2007 11:42 am

It could be. The usual explanation for a low A260/A280 is protein contamination, which would suggest that your phenol extraction wasn't efficient. You won't be using all your prep to do a pcr, so you may as well try it. If it works, who cares why you have a low ratio, right? If it doesn't work, you can always repeat the phenol extraction in the proper sequence and do the pcr again.

soso
Garter
Garter
Posts: 12
Joined: Tue Mar 27, 2007 3:32 pm

Post by soso » Mon Apr 23, 2007 12:51 pm

Hi..so what did i know is that phenol remove all protein so whats the exact action of the chloroform?? is it to remove the phenol??..
thanks

blcr11
Viper
Viper
Posts: 672
Joined: Fri Mar 30, 2007 4:23 am

Post by blcr11 » Mon Apr 23, 2007 5:37 pm

Yes. The phenol extraction removes the protein and the chloroform extraction removes any residual phenol left in your aqueous phase. Some protocols even do two chloroform extractions after the phenol step.

Post Reply

Who is online

Users browsing this forum: No registered users and 9 guests