Blunt end ligation
(by ucgatan from ucl.ac.uk)
Wed Feb 14 08:26:25 EST 2007
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
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 Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson from ucl.ac.uk
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