problem in ligation

[shazia]

Im trying to clone a 2.2kb insert into a4.2 kb expression vector.Im cutting the vector with ncoI which is a cohesive cutter and smaI a blunt cutter.Im digesting the vector with the two enzymes sequentially.The insert is having ncoI overhangs on one side and the other end is blunted using Klenow fragment.Ligation is done overnight at 22degrees using fermentas T4 DNA ligase.After transformation in E.coli DH5alpha I only get self ligated colonies of vector.Both the enzymes are working fine.Individualy they both linearize the vector.I have been trying this for months but everytime i get the same result.Could anybody tell me where am i going wrong?

[Warthaug]

You're probably loosing BP's off of your overhang, leading to a blunt ligation. Either that, or there is insufficient space between the two restriction sites and as such you're only getting a single cut (many restriction enzymes work poorly unless there is ~10bp from the cut site to the free end of the DNA).

Your options are:
1) Ligate at a lower temp; I usually use 16C, as this seems to avoid loss of the overhanging end
2) Use a different set of enzymes, if you're cut sites are too close together on your vector.

Bryan

[ramnivas]

If you are doing ligation for overnight then 16C is best temp for it. and check the star activity of enzymes. and try it again with provided specifications or you can do double digestion before ligation.

[alichem]

Try sequential digestion

[shazia]

Thanx for ur reply.The two RE sites are spaced at a distance of 20bases.So i dnt think there is any such problem.I can try by changing the ligation temperature.Wud let u knw soon.

[Moleecule]

[Only registered users see links. ]
Usage notes:

1. Klenow Fragment (3´→ 5´ exo- ) is not suitable for generating blunt ends because it lacks the 3´→ 5´ exonuclease necessary to remove non-templated 3´ additions.

What kind of insert 3' end are you trying to blunt- what type of overhang does it have?
[Only registered users see links. ]

Also digest with NcoI AFTER blunting the other end.

Good luck!

[scrat]

How long are you digesting the vector for?