Subcloning large PCR products

Wed Jan 31 12:11:54 EST 1996

In article <1996Jan29.194614.8132 at mcrcr6> , rosasg01 at
>Subject: Subcloning large PCR products
>From: rosasg01
>Date: 29 Jan 96 19:46:14 EDT
I have successfully cloned 7, 9 and 12Kbp fragments made with the
Expand PCR kit. HOWEVER, I spent months trying to get the first two in.
Blunt cloning into EcoRV of pBSIISK, T-tailing into pGEM-T, self-ligating
the product (which had only one site engineered into one end) followed by
(SalI) and cloning into pBSIISK. Blunt cloning finally worked but I do
not know
why (sorry) and I got very few positives (only one for the 9Kbp fragment).
This pissed me off, so....

For the 12Kbp fragment I built AscI sites into the primers. AscI can cut
very efficiently right up to the ends of DNAs and it is a GC rich 8 cutter
and cuts rarely. I took my PCR product and cut it with AscI. I then
cloned into
AscI cut pNEB193 (NEB) or MluI cut (sites are compatible) pGEM5. BINGO!
clones right away. Maybe I was just lucky this time but give it a try.

Good luck
Mike Dalrymple

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