Transformation and ligation problem - Trasnformation of ligation sample shows only vector size.. (Jun/30/2010 )

Why does transformation of ligation sample shows only vector size after digestion of plasmid prep of the transformant?

Are you saying that you don't see any insert release ? If all colonies show the same size then your ligation didn't work

This could be because maybe your plasmid religated to itself. or your PCR product didn't digest well to ligate to your digested plasmid. this happened to me many times.

Did you do double digestion? or TA cloning?

biosys on Jun 30 2010, 01:03 AM said:

Curtis on Jul 1 2010, 05:05 PM said:

Many thanks for your reply.

Yes, I don't see any fragments released after double digestion by KpnI-XhoI (Fermentas) but when purified the insert fragment from another recombinant it is clearly visible with correct size before ligation. After ligation @4 degree overnight and transformation in DH5@, I got some colonies but after digestion, no insert fragment but only the vector size.. Is there possibility of self ligation of vector?? if so, how to overcome that problem??

So, let me see if I've got this straight...

The insert you're trying to clone was liberated from another vector by double digestion with KpnI and XhoI. The vector you're trying to now re-clone this insert into was also digested with KpnI and XhoI.

Is that right?

If so, then it seems your ligation didn't work (as Curtis suggests). The source of your vector-only transformants is likely undigested vector in your ligation mix.

Did you gel-purify both your liberated insert and your double digested vector before combining them in the ligation mixture?