No Colonies on subcloning - (Apr/13/2006 )

Cutting with a single enzyme and not CIP treating should result in tons of colonies if the ligation worked (many, perhaps most, will not have an insert, but you should get colonies. Since you're not getting anything, it must either be:

1. The retriction digest is not producing compatible ends, perhaps due to star activity (but I would still expect some to be cut correctly and thus ligate and produce colonies).

2. The ligation is failing (perhaps you're misinterpreting your ligation mixture gel?).

3. The insert is toxic to your host strain (unlikely, because you were able to TA clone it, but perhaps in the TA clone there is no promoter, or the insert is in backwards. I would still expect colonies in your cloning step arising from oppositely oriented inserts, but perhaps not).

4. Your cells are not competent enough (but your positive control is working).

5. Your media or selection strategy is incorrect.

6. The fragment you've recovered from the TA vector is not what you think it is, and perhaps contains an origin of replication that is incompatible with your final vector.

7. Something in the ligation mix is inhibiting transformation (perhaps you should precipitate your ligation mix?).

There are really no other possibilities that I can come up with, so one (or more) of them must be the problem. Have you tried a ligation control -- just linear vector plus ligase?

Can you tell us exactly what you've done so far, starting from the PCR? What TA cloning vector did you use? What is the size of your PCR product? What enzyme did you use to liberate the TA cloned insert? What enzyne did you use to linearize your final vector? Are there restriction sites engineered into your PCR primers?

If I had to bet, I'd say your ligation is failing, regardless of what your ligation gel is telling you. If the ligation were working, you'd expect to get a ton of vector-only transformants if nothing else, and your not getting them. Most of the other possibilities are really just theoretical possibilities...

Do the vector only ligation control. If you can not linearize and then recircularize the vector alone, you'll never get your clone.

How and when did you prepare your competent cells? When you say your postive control is working, what does that mean? For example, if I put 1 µl of uncut vector into 500 µl of competent cells and plated them, I'd expect a solid lawn of transformants, even if I plated at moderate dilution. Is this the kind of result you're getting, or are you getting isolated colonies?

hey Folks,

Finally, I got Clone...
I should say its by God's grace

Its interesting, i had the same bands in ligation

i did not CIP my vector,
-ve gave me 17 colonies
10:1 ratio gave me 35
3:1 gave 3

and out of 15 screens i got one which had my insert...

Re- Search n Re n Re n Research i guess...

Thanks to all of you for ur response.