ligation problem - self ligation of vector - (Mar/10/2005 )

Hi, I'm making a ligation of a 1200 bp insert into a pECFP-N1 vector but the vector always makes probably a self ligation! My finding is the next:
I digested the vector and the fragment with two different enzimes (HindIII and BamHI), and found that double digestion works well and sticky ends are not complementary. Then the fragment is ligated into the vector according to a conventional protocol. transformation was performed wirh the ligated product and also with the double digested vector as a negative control for checking the self ligation and after plating i foun the same number of colony on both the plate of the ligation reaction and the plate of negative control. The same problem happened to one of my colleagues using the same vector but different restriction enzimes (EcoRI and BamHI).
Every suggestion will be wellcome!!!
thanks in advavance for your help.
Have a nice day and a good work!

hi
you can find an helpful discussion at this topic :

http://www.protocol-online.org/forums/inde...?showtopic=5292

In your case, i strongly recommand you to dephosporylte your vector and to phosphorylate your insert.
Moreover increase the relative quantity of insert than plasmid. Usually i use 5fold more insert mass than mass of plasmid. But once i was forced to use up to 10fold...
Fred

Thanks for your suggestion, but after dephosforilation no colonies growth in the control and in the sample!
But the real problem is that i had just made a ligation with this vector and with the same restriction enzymes two months ago and the control (digested vector with ligase and without insert) gave me only 5 or 6 colony!!! We can't understand why now the vector make a so high self annealing even if it hasn't compatible sticky end.
Are there other control we can perform???
Thank you for every suggestion
Have a good day and a nice work

hi
did you phosphorylate your insert?
after precipitation of the ligation mix (Na ac, tRNA and etoh) i usually resuspend in 10µl and use up to 5µl for electroporation. I know that if bacterias are chemically competent the efficiency of transfection is not as good as electroporation....
Maybe can you use more of ligate in order to transfect.

after electroporating, it 's recommended to let your bacterias grow for 30' to 1hour. You may increase this time?

Hope that helps you.

Fred

I tried a lot of different ligation test changing the ratio of insert and vector, the time and the temperature of ligation, the dephosphorilation of vector and perform not a duoble digestion but two different digestion but I always obtain a self annealing of the vector and no positine ligation product.
I don't have idea if there are some other protocoll I can perform...
should be a good idea trying a ligation whit a low melting point agarose???
I'm in a huge empass, and so every help is well accepted
Thanks to everyone
Have a nice day

it sounds to me that you're digesting your vector incompletely, where only one RE is actually cutting, which will explain why you have high number of control ligation colonies and why you don't get your insert into the vector.

make sure that you are actually creating the two restriction ends. There is no way to verify this simply by running your doubly-digested vector on a gel (I assume that the two sites are on the MCS and within close proximity).

With an EcoRI-BamHI double digest, it should be done in the EcoRI buffer according to NEB Catalog. The only way I can think of checking for RE activity is to do single digests in EcoRI buffer for both enzymes, and check for linearization, which is detectable in the gel (run an uncut vector on the lane beside it).

There should be no need to dephosphorylate your vector.