Geneclean

Carol Enderlin enderc at essex.hsc.colorado.edu
Wed Jun 16 22:00:29 EST 1993


I have a question to ask, and a method suggestion to pass along.

1.  First the question:
What's the largest (and smallest for that matter) fragment that
bionetters have purified by geneclean and successfully cloned.
I'm asking because I wanted (note past tense suggesting less than
success in first tries) to subclone large fragments from cosmid
clones.  My two fragments are slightly smaller and slightly larger
than 20 kb, respectively. One came out as a complete smear, the other
had less smear, more of a band, but didn't look quite as large as
the starting band in the digest.

To avoid the obvious suggestions, I'm now trying low-melt agarose
(I just love geneclean, and am really dissatisfied with the way
low-melt gels run) to get the fragment. I am also looking at 
figuring out smaller fragments to clone to avoid cloning these. 
However, this makes me wonder if people have had success with 
10-15 kb fragments.

Email, or post replies to this newsgroup. I will 
post a summary. Let me know if you wish to remain anonymous.

2.  Now, for the suggestion. 

This comes partially in response to a question posed a few weeks
back about geneclean and ligations. I will clip part of the question
below for clarity. The suggestion is a method that has been passed around
here in Denver between National Jewish Hospital and the University
of Colorado Health Sciences Center. 

For the purification of small DNA fragments using geneclean kits:
(Because recovery of 200-500 b.p. fragments can be fragment 
dependent, some work fine whereas others don't)

Gel piece in tube. Follow directions for how much NaI to add.
Then prior to melting (not sure when is critical) we add
70 ul of 0.5% Acetic Acid per 0.5 ml of NaI solution. This
makes the pH approximately 6. Proceed as usual. The solution
changes color a little bit (I'm usually using colored tubes,
but it appears to turn yellow).

The post I'll quote below made me think I should post this
tip, however, I do not address the issue of inhibition of
ligation and he does not address the issue of fragment recovery. 
However, if you have better recovery of fragment the ligation may work
adequately.

> From: Abush at watson.princeton.edu (Andrew Bush)
> Subject: geneclean hassles
>
> I have had variable success with Geneclean and I wanted to find out a) if
> this experience is mirrored out there in molecular biology land b) maybe I
> am doing something wrong. Is there any step in purifying DNA on glass milk
> that is particularly prone to problems?  I follow the geneclean protocol
> carefully and I have even gone back and read the original paper describing
> glass-based purification of DNA (from Vogelstein's lab).  

> I am generally using this method to purify inserts (fairly small 0.3-2 Kb)
> and what I see is a clear reduction in the number of colonies (amp
> resistant)  in the insert as compared to vector only.  I do not see this
> effect with inserts purified by Low-melt agarose protocols.  So, I assume
> that there is some kind of inhibitor in the geneclean preparation. 

Carol Enderlin
enderc at essex.hsc.colorado.edu




 



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