2 replies to this topic

[bumbilko]

Hi all!
I am stuck with cloning of DNA from an environmental sample. I hydrosheared my DNA (instead of digesting it with restriction enzymes) in order to obtain completely random fragments. Then I used end repair kit to obtain blunt ends suitable for cloning into the blunt vectors provided by the same company. But I obtained only 5 colonies plating 50 microliters of the transformats. Transformation control works well, but the ligation control gives me just 25% of the expected number of colonies. Therefore I think the problem must be in ligation. I also tried to use different amounts of insert DNA (to change insert:vector ratio), but I am allways ending up with just 5 colonies.

I have already read on this forum, that blunt end cloning is very difficult...(( But I would like to continue with my idea to produce random fragments by hydroshearing.

Do you have any idea how to improve my ligation reaction?

Edited by bumbilko, 10 February 2011 - 09:10 AM.

[Curtis]

not always blunt end ligation is difficult. I use pJET vector from Fermentas. it works really great. in fact I like it more than TA cloning and stuff...

[Adrian K]

Curtis, on 10 February 2011 - 08:19 AM, said:

not always blunt end ligation is difficult. I use pJET vector from Fermentas. it works really great. in fact I like it more than TA cloning and stuff...

Yup, I do agree with you Curtis, I do use the same product as you (it is a trial kit given to me). pJET works great.

bumbilko, the cloning efficiency are probably due to your competent cells, ratio of fragments with vector and the quality of the template. Either you had put in too much template, or your template is yet to be completely sheared... but I have to admit that I have not used any hydrosharing method before.

Edited by adrian kohsf, 10 February 2011 - 09:23 AM.