22 replies to this topic

[tuhin_genes]

mario2004, on Mar 13 2005, 08:50 PM, said:

Thanks Nick. Very helpful.

I still want to know how much ethanol should be used for washing.

Mario

well it depends but generally i ml should be sufficient to wash the DNA pellet. try to aspirate the ethanol carefully...add i ml of 70% ethanol again...centrifuge briefly and aspirate the total ethanol and then if possible go up to the dessicator for remaining ethanol if any. keep it for 30 mins or so... i hope this helps.

[s_laub]

I just started working in a lab, but I've always been using 300 ul of 70% EtOH, followed by 300 ul of 99% EtOH after a miniprep and my supervisor said that's fine. But the preps are obviously dirty because they can't be cut with REs. So should I always wash with a minimum of 1 ml?

[swanny]

s_laub, on Sep 7 2009, 02:23 AM, said:

I just started working in a lab, but I've always been using 300 ul of 70% EtOH, followed by 300 ul of 99% EtOH after a miniprep and my supervisor said that's fine. But the preps are obviously dirty because they can't be cut with REs. So should I always wash with a minimum of 1 ml?

As a few others have stated, the actually volume isn't the most critical issue, so I wouldn't stress too much over it; you PI's advice is probably the one to follow.
Having said t hat, when we had digestion issues, we did one of two things. Once the DAN was resuspended, we put it through a second EtOH precipitation (or did a PCR cleanup on it). This is fine for most contaminants.

If, however, the repeat precipitation didn't do the trick, we went back and recultured, but we used LB. A  few of our group found that overnight culturing with the really rich media gave higher yields of cells and plasmids (no surprise there!), but the DNA quality was lower. Growing cells in LB gave lower yields (fixed by larger cultures!) but better digestion.

[s_laub]

Interesting! I'm using YT, which according to my sources (http://micro.fhw.oka...media/lbyt.html), is rouhgly the same as LB for a 1 L stock.

Any idea on why a less rich medium would give better digestion results?

[swanny]

s_laub, on Sep 7 2009, 04:13 PM, said:

Interesting! I'm using YT, which according to my sources (http://micro.fhw.oka...media/lbyt.html), is rouhgly the same as LB for a 1 L stock.

Any idea on why a less rich medium would give better digestion results?

Not sure, but I would imagine the answer is somewhere in the area of metabolite buildup, particularly with an overnight incubation. Possibly aeration may play a part.

[janetvelasquez80l]

What percentage of ethanol should be used?

Hubschrauber selber fliegen

[Vini]

infonavit222, on Feb 24 2010, 11:11 PM, said:

i still want to know how. much ethanol should be used for washing.

we use 1ml of 70% EtOH for washing.  But 600 uL would work equally good....idea is to wash the pellet....so such variations, i dont think, matter.........

[Maddie]

Here is a new paper on ethanol precipitation I thought I'd share.
Let me know if you want me to post the pdf somewhere.

http://www.ncbi.nlm....pubmed/20336673