5 replies to this topic

[nirvana]

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.

[jiajia1987]

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

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.

i have had the same problem too. are you using self-prepared competent cells or commercial cells?

[T C]

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

[scolix]

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?

[swanny]

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.

[molgen]

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