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
(by the way, I checked the “cleavage close to the end
of DNA fragments” from NEB page so primers should be
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On Feb 13, 9:20 am, Ricardo <[Only registered users see links. ]> wrote:
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
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?
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!
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) [Only registered users see links. ]
On Feb 14, 8:26 am, Tom Anderson <[Only registered users see links. ].uk> wrote:
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.
Can any body tell, after restriction enzyme digestion, can i directly (without purification) add klenow fragment to make blunt end of the insetr? Accoriding to neb protocol, Klenow fragment is active in any restriction enzyme buffer.
One more question, Can I add directly CIP to the digested plasmid without going any purification treatment? According to neb protocol CIP is also active in neb buffer 1, 3 and 4