request follow-up on SmaI (PCR subcloning woes cont.)
txpljfg at UABCVSR.CVSR.UAB.EDU
txpljfg at UABCVSR.CVSR.UAB.EDU
Wed Mar 9 11:19:02 EST 1994
I apologize for not posting a followup on the sma problem. Shortly
after the replies were received, a bug in my mailer software blew away
the message store and I lost all my saved messages. Fortunately, I had
printouts of everything.
My history of cloning was almost exactly the same as yours. I used
a couple of batches of blunt-ended smaI cut pUC19 for years and
produced a number of libraries from PCR amplified material with no
problem. It was so easy I couln't understand whay anyone else was
having a problem.
Then I got a real job and started my own lab (with a new batch of
enzyme). AT that point I could not clone *anything*. All of a sudden
a trivial, no-brainer procedure becasme impossible. Like you, I wasted
at least a month trying to clone stuff. Rounding up the usual suspects
I received a total of three replies: The first one simply described a
way of working around the problem by screening the bacterial colonies.
This, however, did not address the cause, and my problem was too severe
for it to work anyway. The other two replies suggested that I had a
bad batch of sma I. Some batches of smaI are contaminated with an
exonuclease that will remove some bases from the ends. This was, in
fact, the problem. I sequenced a number of white colonies that did not
contain inserts and found that 2-3 bases were removed from the cut
site. I tried several batches sma and found that the backrounds did
vary (2-30% - sound familiar?). When I used batches of smaI with low
background, the problem went away. I found the best batches of sma
were from Gibco/BRL (so was the *bad* batch) and B-Mannheim. As a
further hedge, I now incubate with the minimum amount of sma I that
will do the job in 1 hour.
I have sinced stopped using blunt end cloning and I now use TA cloning
for all my PCR products. I incubate blunt-ended pUC19 with 2mM dTTP in
standard 1X PCR buffer for three hours at 75 degrees, ppt with ammonium
acetate and isopropanol, wash a couple of times and ligate normally.
Good luck..Let me know what you find.
> When during November last year you posted to bionet.molbio about your
> hassles with SmaI, I recall being slightly interested because of some
> difficulties I had with a blunt end library cloning run.
> I gophered the archives just now to prompt my memory. The reason?
> I have just lost about a month in trying to do a simple (for me)
> cloning experiment using pUC19/SmaI. In your original post you stated
> how the background phenomenon was not host dependent (I concur) and that
> you had a general history of success. Same here. I've never found blunt
> cloning difficult over about 7 years and have never resorted to linkers
> in that period. Batch preps of PUC19 and Bluescript cut with SmaI have
> served me well since 1987 with almost zero background. The enzyme was NEB's.
> But wouldn't you know it...I have now tried two batches of NEB SmaI,
> (one old, one new) and from Toyobo, with different buffers and I get
> backgrounds ranging from 5 to 30 %. (I ran out of the original batch
> of cut pUC19). The purified pUC19 is OK since I can cut with HindIII
> and get no background, with the same ligase as used for the dud results.
> I have repeated this digest about 6 times. Phenol extraction doesn't
> change the situation. Clones that have inserts seem to be OK with the
> correct CCC-GGG ends.
> Do you have any follow-up results that could help my predicament?
> Andrew Healey
> email: andrew at qimr.edu.au
> The Bancroft Centre, Queensland Institute of Medical Research
> 300 Herston Road, Brisbane, AUSTRALIA 4029
> O'Reilly: Just remember, Mr Fawlty, there's always someone worse off
> than yourself.
> Basil: Is there? Well, I'd like to meet him. I could do with a laugh.
James F. George, Ph.D. "Back off man, I'm a scientist"
Department of Surgery --Bill Murray
University of Alabama at Birmingham
txpljfg at uabcvsr.cvsr.uab.edu
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