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.