Blunt end ligation

[peter]

On Feb 14, 8:26 am, Tom Anderson <ucga... from ucl.ac.uk> wrote:
> On Tue, 13 Feb 2007, peter wrote:
> > On Feb 13, 9:20 am, Ricardo <nightfall... from yahoo.com> wrote:
>
> > > 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)
>
> > 1. AP does not inactivate easily, run trough a gel instead.
>
> Or perhaps just do some sort of cleanup - i'm doing a blunt-end ligation
> at the moment, and after the Klenow and AP steps, put the products through
> a QIAquick column. I reckon getting rid of all the leftover enzymes, salts
> and whatnot gives my ligation the best chance of working (i'll let you
> know in a few days if i got any inserts ...). A phenol-chloroform
> extraction, or maybe even just an ethanol precipitation, would probably
> work just as well.
>
> If you run the stuff on a gel, you have to do some sort of cleanup after
> that anyway - is there any advantage to having the gel step as well?
>
> > 2. Circularization of the insert is not a problem, since insert does
> > not have origin of replication.
>
> True. There is a risk of concatamerisation of the insert, though; i've
> never seen it myself, but i've heard of it happening. I don't know if it's
> just an urban myth!
>
> > 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).
>
> Indeed - the OP should be able to skip the digestion and fill-in steps and
> go to blunt cloning straight from the PCR product. Provided that he's
> using a proofreading polymerase, that is - if not, there'll be an
> overhanging A on the PCR product that will need dealing with.
>
> tom
>
> --
> Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
> (t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.ander... from ucl.ac.uk

Tom,
1. CIP and BAP are pain in the butt to get rid of (phenol is also not
100% ), always use dilluted AP () 1:10 or more) and check the ends for
damage. I always run gel and Qiagen or BIO101 extract. You need to run
gel anyway to remove the uncut vector. Dont watch too long though on
the UV - every second reduces the vector quality by half.
2. even in Taq Polymerase PCR there is about 30% without overhang,
provided that you skip the final extension.
my 2c
Peter