davesmith at bioch.tamu.edu
Thu Aug 15 18:00:02 EST 1996
Carolyn Hoyal-Wrightson <choyal at scripps.edu> wrote:
>Anybody out there ever try the "Seamless" cloning method from
>Stratagene? It sounds like a great system but somehow company product
>literature can make anything sound good. I was just wondering if anyone
>had noticed any quirks, or if people have had good luck with it.
>The Scripps Research Institute
I've used the Stratagene kit twice with acceptable results.
I have four constructs that I'm after. On the first go-round I
saw very little PCR product ( their protocol is based on only
15 total rounds of amplification with Pfu pol). Forging ahead (after
talking to a tech rep who said "don't worry about it!"), I got one of
the four clones. On the second run (from scratch) I got two more of
the desired clones, for a total of three out of four ( I tickled the
PCR with Taq--a reverse trick of Stratagene's Taq Extender). I have
sequenced my gene in two of the three constructs and found no
errors--there still may be errors elsewhere on the plasmid. (To get
rid of this caveat subcloning from outlying sites into a good vector
backbone will be necessary. )
I've had problems with "quantitiy" of PCR product. I believe this is
due to two factors, mainly-- 1) Pfu is not as hearty as Taq--a
sacrifice for fidelity, and 2) supercoiled DNA just doesn't amplify
like linearized DNA. If you have a unique restriction site in your
gene (and I haven't until yesterday), it should be a sure thing.
I highly recommend the kit and suggest linearizing your vector for the
BACKBONE PCR product. With clean PCR product in hand, you can have
possible clones the next day. There's no purification of restriction
fragments and the ligation is performed at room temp for 30 minutes.
The kit comes with X1Blue supercompetent cells.
If they get the kinks (poor yields, etc) worked out of this system, it
will revolutionize molecular biology.
Best of Luck!
--no association with Statagene whatsoever---
...just my two bits.
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