Unstable/Loose protein interface

[molecular_micro]

Hi,

I am new to much of this molecular work...I have previously worked on applied microbiology projects.

I have been performing a fairly standard phenol-chloroform extraction but have hit a snag. Following cell lysis I am treating cells with 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuging (15,000xg for 10 mins). The resulting suspension is separated with a clear aqueous layer and dense organic layer. However, when attempting to aspirate the aqueous layer for precipitation the interphase layer is extremely loose, to the point you cannot aspirate the aqueous layer without disturbing this interphase.

Has anyone else had similar problems? If so, how would one rectify these?

Thanks in advance for your comments

[mchlbrmn]

Depending on how much material you think you have to spare, you can not take all the aqueous layer, but leave the interphase undisturbed and lose some of the supernatant (Is that possible in your case?) or you can do the same, but then do a second back-extraction adding water (or Tris EDTA), vortexing again (or not sometimes if extracting high molecular weight genomic, but repeat however you did the extraction), spinning, and taking 80 or 90 % of the supenatant to lose only .2 x .2 = 4% of the total (if only 80% recovered each time). Just make sure that if you do the back extraction, that you take into account if you have diluted the supernatant, so for example if the subsequent step is precipitation you may need to add extra salt (as sodium acetate or sodium often, or NaCl if that's not convenient) to precipitate efficiently.

(Another possibility might be to spin the solids out of the solution before extraction, if this can be done without losing the DNA.)

If the lysate solution is so viscous that when you pipet it off the organic phase, the interphase that you are not touching with the tip is disturbed and pipetted up by viscous goop stirring it up, it may be that the DNA is too concentrated, too viscous, and needs to be diluted in more lysis buffer before extraction is attempted.

[relaxin]

You may need to sacrifice some of the aqueous phase in order to avoid getting the interphase. If you accidentally pipet some of the interphase or organic phase with the aqueous phase, transfer the mixture into a clean 50 ml conical polypropylene centrifuge tube and centrifuge for 5 min. Now the small amount of organic phase will be at the bottom, you can pipet out the top aqueous DNA solution with little contamination.

[molecular_micro]

Thanks for your reply,

with regards to taking a small amount of sample, I was able to aspirate <10ul before the interphase was disrupted, so I was recovering negligible amounts.

With regards to spinning out the solids, this would be performed between lysis and phenol:chloroform step? I would then retain supernatent and continue with the extraction.

I'm interested by your final comment, I performed a protocol as per publication (but we both know that published methods do not always give the nitty gritty)...The lysate looked extremely 'thick'/'gloopy' prior to addition of PC, are you suggesting that my DNA conc. is at such a level that it is pulling up (in want of a better description) the interphase proteins?

Thanks again for your input here...I've got a conference in a couple of months and these molecular biology problems are causing me a real headache in terms of getting results for my poster.

[mchlbrmn]

With regards to spinning out the solids, this would be performed between lysis and phenol:chloroform step? I would then retain supernatent and continue with the extraction.

Yes, that is what I meant, but I'm not really sure it's good advice, especially since I can't see what you are doing. As I mentioned I might be concerned of losing the genomic if it's still stuck to the proteins, although I think this might help. It is listed as an alternative in RNA guanidinium extractions. It may be safter to spin it out after the extraction, as Relaxin suggested.

The lysate looked extremely 'thick'/'gloopy' prior to addition of PC, are you suggesting that my DNA conc. is at such a level that it is pulling up (in want of a better description) the interphase proteins?

What has happenned to me, on occasion, is when the lysate is very viscous while I pipet up the supernatant with my tip well away from the interphase, the tip grabs a piece of heavy, viscous "snot" that whips up the interphase at a distance from the tip, pulling solids up into the supernatant even though my tip was not near the interphase.
Is this a genomic DNA extraction?

[molecular_micro]

Yes, I am extracting genomic DNA from Listeria monocytogenes. From your description and following a quick chat with my supervisor I think you may have been correct in your suggestion that the DNA is excessively concentrated. I will decrease the starting cell count prior to lysis and then if necessary I will add TE buffer to my lysate, prior to PCI, this will dilute down the DNA and should allow removal of the aqueous layer.?

Thank you both for your suggestions/advise, guess I'll see if I can get this working tomorrow.

[relaxin]

I have a feeling that you did not do a Proteinase K digestion before your phenol-chloroform extraction. The genomic DNA is so intact that it will be difficult to pipet the aqueous phase out without disturbing the interphase.

If you mentor can afford it, I would suggest you to use QIAGEN Blood & Cell Culture DNA kit. It does not use phenol-chloroform extraction.

[molecular_micro]

Hi all,

thanks for all of the feedback. Just a note to say that I finally got some good quality, high yield DNA I underwent a double PCI step (with back extraction)...removing a fairly large amount of the protein interface during the first extraction. I then undertook a double chloroform step, I know its long winded but finally got the downstream PCR going...So thanks for all of the advise.