Confusing transformation results. - (Aug/01/2007 )
I am trying to ligate a 4.9kbp fragment into pTK.
I have digested both with XHoI/SacII, run on a gel and purified. I dephosphorylated the vector using SAP and then used T4 ligase at 16 C overnight.
I used TOP10 cells for my transformations and used Xgal.
There are no blue colonies at all (so I was thinking maybe the vector doesn't actually have lacZ -any ideas how I know if it does or not?) but also there are a lot of colonies on all plates including the control plate (vector but no insert) I'm not really sure what this means. Does this mean my plasmid is religating to itself? how can it do this when I have digested producing two different sticky ends and SAPed it?
Any suggestions would be greatly appreciated on how to get this to work! Thanks
did you amplify your dna fragment or cut it from another plasmid? If you amplified using PCR you will need to have incorporated 5' phosphates on your primers.
If you didn't gel purify your cut vector, you'll have circular plasmid carry over from the digest and modifications. This will show up on you vector (no insert) controls.
ian
this can happen if the vector is not completely digested by both enzymes. You would have to digest for longer and then run the gel to purify the vector.
So I redigested for longer and with more enzyme and then did SAP but still lots of colonies on the control plate. Is there anyway I can check if my SAP has worked properly? Someone suggested to purify after SAP instead of before, is that preferable? Could this make a difference?
If you still get colonies in the ocntrol plate, could be that one of the enzymes is not working properly.
Try digesting with both enzymes separately and run it on gel to verify if both enzymes digest.
We usually heat inactivate after SAP treatment. This is usually fine and one can continue with ligation without purification again.
Try digesting with both enzymes separately and run it on gel to verify if both enzymes digest.
We usually heat inactivate after SAP treatment. This is usually fine and one can continue with ligation without purification again.
Firstly, to check whether your plasmid has blue/white selection, plate the uncut vector on X-gal under conditions that induce the promoter and you should get blue colonies. If you don't then it is likely that pTK does not have the LacZ alpha fragment so can't be used for this kind of screening. Are you sure the ligation hasn't worked? How many white colonies do you have on the ligation and ligation control plates?
To test whether re-ligation of your vector is the problem, perform the ligation with vector only (no insert) both with and without ligase. If there are more colonies in the ligase-containing reaction then the problem is re-ligation.
I would agree with Scolix's suggestion is that the most likely explanation is that the vector is not being fully digested. Do as Scolix suggests - make your preparative (double) digest and in parallel perform single digests. Run all of the digests on a gel along side an uncut sample of your plasmid to check that both enzymes are cutting properly.
Assuming that both enzymes are cutting fully, ensure that before cutting out your preparative double digest band you run the gel for as far as possible e.g. until the vector band is 3/4 of the way down the gel. The further you run, the more separation you will get between digested and undigested plasmid.