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cloning/ligation problem? (shRNA into vector) - cloning, colonies in neg control (Mar/21/2007 )

Hi everyone!
This is my first time cloning and hence having a hard time troubleshooting. I have been trying to clone shRNA into pSUPER vector and have been running into a bunch of problems (different problems everytime!!!). This time I have got colonies in my negative control (vector alone with no insert). My protocol was: double digest with BglII and HindIII, ligate o/n @ RT (T4 DNA ligase), inactivated @ 70 deg C for 10 min and used the reaction (5 microliters) for transformation in DH5alpha. Now I have colonies in all my groups INCLUDING my negative control. How does one go about after this? Do I just pick colonies and go to the next step or I start all over from square one? Any ideas/inputs will be highly appreciated.


You have to digest the vector for longer and run them on a gel long enough to seperate the uncut from the digested one. Having colonies on the control means the vector is not digested competely or there is uncut or single cut plasmid.


Dephosphorylation of doubly-cut plasmids is useful for preventing the self-ligation of incompletely singly-cut plasmids. A neat trick mentioned by one of the forumites in a post some while ago is to cut the ligation with a restriction enzyme found in between the two cloning sites in the MCS (but not in the insert!) in order to linearise re-ligated or undigested plasmids and improve the transformation efficiency of correctly ligated plasmids.


Thanks for your answers...I will let you know how it worked out!!


To molbio_dc' ,
I am trying to transformation on JM109 using psuper retro puro..I used same protocol as in the manual as well as ligation with increased insert vector ration of 5:1. But still I am not able to get the transformed colonies. In negative I have got lot of colonies. For Ligation I could not use the exact protocol as the manual because I had my gel cleaned vector concentration very less. So I used 8 microlitres so that it will be around 80ng of vector and for that i calculated ng of insert needed and added.
Please help me.