Blunt end ligation

[peter]

On Feb 13, 9:20 am, Ricardo <nightfall... from yahoo.com> wrote:
> Hello, i am having a problem doing a ligation with a
> PCR product with the restriction sites (sticky ends)
> on the primers. So, as i think the problem might be in
> the digestion, i decided to do a blunt end ligation
> using the same primers, digesting them and fill it
> with klenow. As i never did a blunt end ligation
> before i would like to see if you can find any problem
> in the protocol i made for this procedure:
>
> (primers for PCR will be phosphorilated to do this)
>
> 1. Digest of vector and PCR product with xhoI and ndeI
> (both originate sticky ends)
> 2. Gel extraction of both
> 3. Make blunt ends with Klenow
> 4. Dephosphorilate the vector
> (And after inactivate the AP 65 degrees for  15 mins)
> 5. Ligation 1:5 with 5% PEG (final concentration)
>
> This insert size (PCR product) is about 1kb and the
> vector about 6kb.
>
> My worst fear here is that, since the insert is big
> enough, it might recircularise, even with PEG.. I
> already had all kinds of recircularization, even with
> one sticky end and one blunt end!!! so i am a lil
> traumatized with that. Can you please check if this
> procedure is alright and if you have any further
> advice to this i would appreciate it.
>
> Ps: I know i could do topo cloning but i would rather
> not.
> (by the way, I checked the "cleavage close to the end
> of DNA fragments" from NEB page so primers should be
> alright)
>
> Ricardo
>
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Ricardo,
1. AP does not inactivate easily, run trough a gel instead.
2. Circularization of the insert is not a problem, since insert does
not have origin of replication.
3. If you can use Sma or EcoRV in your vector you could save some
aggravation by using "forced  cloning" strategy (even without
digesting PCR).
my 2c
Peter