Ligation Problem - Molecular Biology (Feb/04/2006 )

I have ligation problem. My insert is 775 bp and I have to clone it into pET32 vector.
I digested my vector with EcoR V (blunt) and Hnd III (sticky) 37 c incubation for 90 minutes and heat inactivation at 80 c for 20 minutes and dephosphorylated vector with SAP at 37 c 60 minutes , 6 ul Buffer 3 ul enzyme ,1 ul water in 40 ul digestion mixture and after it heat inactivation at 65 c for 15 minutes.
Then gel purify my vector and insert (not dephosphoyrlated).
Gel photos are attached.

My ligation misture is 1 ul vector
3ul insert
1ul T4 DNA ligase
1ul 10x buffer
4 ul H2o
after transformation into DH5 ALPHA

20 minutes on ice after ligation mixture transfer to competent cell.
42 c for 30 sec heat shock and
2 minutes on ice
1 ml lb media
37c shaking for 1 hr
but i get nothing even in my control
please suggest me some tips for getting clone

I run my 1 ul ligation on 1 percent gel and result is attached.
my insert in 775 ba and vector i hope 5900 bp.
Please look at suggest me some tips.
first two run is ligation mix 1 ul and thirld is my contol
with out insert

Hi,
When having problems with ligations I usually test to check first if the ligase is OK i.e. cut a vector with just BamHI and see how many colonies you get. I have had batches of ligase from some suppliers that don't work at all, that are useless after a few months in the -20C freezer and some that work fantastically well all the time. Usually the ligase from Promega or NEB is pretty good. Also ensure that you freeze the ligase reaction buffer in small single use aliquots, as the ATP and DTT it contains degrades after a couple of freeze thaw cycles. Ensure that you mix it very well to dissolve the DTT before aliquoting. Other things to do is to NOT dephosphorylate your vector, sometimes CIP or similar treatment works really well i.e. no backgroud self-ligations, other times I find it has completely prevented all ligations. I have triead using phosphatases from calf intestine, shrimp and antartic phosphatase from NEB, but usually dephosphorylating your vector is not worth the bother, I would rather have a high background of self-ligations than no colonies .
For ligations I usually do a digest of the vector (3-5 ug) with one of the restriction enzymes (10 units should be plenty) for a very long time e.g. 4 hours or even overnight, then gel extract the vector and cut with the second enzyme for a long time and then gel-extract again. This will give you pretty much 100% cut vector. For the insert I usually do long digests as well but only gel extract after the 2nd digest. If you prepare your insert via PCR make as much as you can, I usually pool 4 or 5 100ul PCRs to make sure I have loads of insert.
Before doing the ligation measure the concentration of your vector and insert, then to 100 ng of vector add 3 molar equivalents of insert, e.g if your vector is 3 kb and your insert is 1 kb you will use 100 ng of vector and 100 ng of insert. I usually do a 10 ul ligation using 1 ul of 10 X ligase buffer and 1 ul of T4 DNA ligase. Setup the ligation in the morning leave at room temp till the evening and add the entire mix to 100ul of competent cells and transform. I usually get somewhere between 10 to 100 transformants on the vector control plate and 2-5 times as many colonies on the vector + insert plate. The number of transformants seems to depends on the vector, pMAL and pGEX usually give me a few whereas pRSET or pBluescript give me loads.
Obviously this method is long winded in terms of preparing the insert and vector however if you are only doing the occasional bit of sub-cloning then it is worth the effort. Using this method I get pretty much a 100% success rate, provided the T4 DNA ligase is working . I have tried using rapid ligation kits blunt-end kits and adding things like PEG to ligations however the results are usually inconcistent, sometimes it works well sometimes not, the long winded-method described above is the only one I find works well all the time.
Hope this Helps
Bye
Mike