Cloning long PCR roducts
ladasky at leland.Stanford.EDU
Thu Jun 19 15:53:22 EST 1997
In article <33A7FB75.3B21 at adelaide.on.net>,
Bresagen <alonie at bresagen.com.au> wrote:
>I have tried any number of techniques to clone long PCR products over
>the last year, and had very limited success.
>I've tried pCR-Script, pGEM-T, and even cutting at internal sites (ie.
>at sites that are 200-300bp in from the primers) doesn't seem to help.
>Probably the most successful approach has been with pGEM-T, but it's
>still hard work to get a clone. These products just seem to be
>incredibly hard to clone compared to normal PCR.
>Has anyone with significant long-PCR experience got any comments or
>The PCR reactions themselves work well. I routinely amplify 6-10kb from
>mammalian genomic DNA with a Taq:Pfu (80:1) mix.
The problem may be the length of your inserts, not the fact that
they were created by long PCR. Once the length of the plasmid plus the
insert exceeds about 10 kB, transformation efficiency falls off rapidly.
I have used restriction digests to subclone 6 kB fragments from lambda
phage into pBluescript (which is 3kB). A colleague of mine succeeded with
a 10 kB fragment but failed with a 12 kB fragment. The strain of E. coli
you use may have an effect -- someone else in this newsgroup will probably
remember the strain of E. coli specifically tailored for the propagation
of fragments abouve 10 kB, but right now it has slipped my mind.
Unique ID : Ladasky, John Joseph Jr.
Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???)
Location : Stanford University, Dept. of Structural Biology
Keywords : immunology, music, running, Green
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