Posted 12 February 2013 - 11:23 AM
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 .
Posted 12 February 2013 - 11:59 AM
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.
Posted 12 February 2013 - 08:04 PM
Posted 13 February 2013 - 07:36 AM
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...
Posted 13 February 2013 - 07:45 AM