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Cloning problem, sticky - blunt ends ligation fail . - (Feb/12/2013 )

Hello everyone,

I've a problem with the ligation of a 650 bp insert in a 12Kb Plasmid . Both of them have been digested with BssHII ( blunting cut) and SwaI ( sticky cut) and then eluited from gel.
So far I've neve obtained any colonies after transformation. The ligase enzyme works perfectly in fact i did the ligation of 1 Kb invitrogen marker for 3 hours at room temperature without any kind of problem. Even all the positive controls of transformation works perfectly. So my doubts are about Ligation reaction.
Now I'm tying to dephosphorilate plasmid and phodphorilate the insert, respectively with a CIP phosphatase and a T4 kinase.
In last ligations the total amount of DNA ( vector : plasmid 3:1) was about 300ng. (maybe too much for the blunt end ligation??) .
I think even that the large size of this plasmid compared to the small insert one could increase the difficulty of this kind of cloning.
Anyone has some idea ???

Thank you for your attention .

-Danbas28-

Blunt end ligations are quite inefficient, try a couple of days at 4 deg C or up to a maximum of 16 deg C for overnight. 300 ng is a lot for a ligation, the usual recommendation is to keep the plasmid below 50 ng.

Large plasmids can be hard to ligate into as you have relatively few ends per ug.

Make sure that it is molar ratios of plasmid:insert you are working with, not ng ratios.

-bob1-

In our lab we do blunt end cloning but with small plasmid. we also use CIP but instead of T4 kinase we use PNK(poly mucleotide kinase)..But it should work fine with T4 kinase also . We face difficulty when PNK or CIP treatment is not proper, we get lot of re-ligation. I would suggest since its a big plasmid, you can try with different ratios of vector and insert.

-neuron-

thank you both ...
today I'm going to ligate newly, after the use of CIP and PNK (Neb) ... The total amount of DNA will be 100 ng ( vector : insert 1:2) ..... I hope the next trasformation will be fine...

-Danbas28-

I'm thinking even to do a double-day ligation, the first at room temperature for the sticky ends and the other at 4 degree ON for the blunt ones ... maybe it will be more accurate..

-Danbas28-