S-O-S Ligation Not Working - No Colonies After Ligation (Feb/12/2009 )

Hi buddies,

I am trying to ligate my insert which is 333b.p. in length with pET21b+ (~ 5.3 k.b. after digestion) vector for protein expression. Both insert and vector are being ligated by NdeI and XhoI.

My ligation is being done @16deg. centigrade overnight. I dont suspect competent cells/Ligase and any of the reagents as my labmates who worked with the same stuff had their desired clones.

My insert and vector yields after gel-elution are not bad either. I got 50ng/ul of insert and 100ng/ul of vector post gel-elution. I've workedout with 1:5 and 1:10 (v:i) ratios twice but with no success.

Now, this problem is very mysterious to me. Can someone suggest any controls/troubleshooting?

Thanks in advance,
nirvana.

nirvana on Feb 12 2009, 02:53 PM said:

Hey

Do you get religations? Are you Cipping the vector (if you start from the vector, its advisable to CIP as you prevent singly cut vector from religating). I generally use a clone to make sure all the vector is completely digested.

If u don't see colonies at all, try Cipping and directly precipitating or eluting the vector, without running on the gel, it helps.

TC

How long did you digest the vector and insert? I would be a little bit cautious so as not to have overdigestion.
I would check the DNA conc. on gel just to be sure.
Could you post your ligation protocol?

Have you tested that both ends of your insert have been digested? Add some ligase to some insert and leave for ~20 minutes at RT then run on a gel. If you don't get a ladder with at least a 3-mer (which will be ~1 kb for you) then one of your enzymes hasn't cut adequately.

A stuped question:
Did you check that the resistance gene fits the antibiotic in the media ?