Ligation

[jamesgoat]

Ligation

• Quote

Post by jamesgoat » Sun Jun 11, 2006 3:11 am

I tried to transform JM 109 with pGEM-T vector (w/ inserts). But when I plated them on LB/Amp/X-Gal/IPTG, there were only 3-4 colonies of each blue and clear colonies coming out. Wondering what has happened. Will it be the ligation problem or JM109? I did positive control and it comes out ok. Does anyone experience this? Thanks in advance.

[oppox]

• Quote

Post by oppox » Thu Jun 15, 2006 11:16 am

It doesnt matter if u get only one colony as long as its a positve one. I got about 15 about half was correct.

[Navin]

• Quote

Post by Navin » Thu Jun 15, 2006 12:03 pm

I did an experiment before involving restriction digestion, ligation and transformation and I had expected many colonies to form but only 1 did. Then my mentor told me (after much thinking) that this most probably occured because the restriction enzyme I used was very inefficient and may not have cut the vector - so only 1 colony formed. So now, I am increasing the amount of the restriction enzyme and I hope that I get many colonies once I transform them.