1 reply to this topic

[priyak]

hi team

            i just started working with cloning....my problem is that i did n't get any transformed colonies my vector is pcr2.1 of 4000bp, and my insert size is 1620bp, digested with Kpn1 and Bamh1. i checked the efficiency of my competent cell,its fine,checked the efficiency of ligase enzyme.its fine

Then i icreased the insert:vector ratio from 1:3,1:5, increased incubation..1hr to overnight. but no colonies....can any one help me in this problem?


Thanks
priyak

Edited by priyak, 26 March 2009 - 07:41 AM.

[rkay447]

priyak, on Mar 26 2009, 09:40 AM, said:

hi team

            i just started working with cloning....my problem is that i did n't get any transformed colonies my vector is pcr2.1 of 4000bp, and my insert size is 1620bp, digested with Kpn1 and Bamh1. i checked the efficiency of my competent cell,its fine,checked the efficiency of ligase enzyme.its fine

Then i icreased the insert:vector ratio from 1:3,1:5, increased incubation..1hr to overnight. but no colonies....can any one help me in this problem?


Thanks
priyak

Your ratio is the wrong way.  You want a 3:1 insert:vector ratio.  This alone could be the reason why you aren't getting colonies.  You need to make sure your quantification is correct by running a small amount of the insert and vector together on a gel.  Compare intensity to a quantification marker.  Take 25ng vector and, according to the sizes you said a 3:1 ratio with 25ng vector would mean you need to add 30.4ng insert.  Reset up the ligation with the proper insert ratios and try it again.