Ligation Problem: Colonies on negative control plate
Posted 10 March 2009 - 06:05 PM
The fragment that is cut out is only 17 bp and it should be in the flowthrough during PCR purification using the QIAquick system.
I did overnight ligation inlcuding a negative control where MQ water was used in place of the insert, 16C using T4 DNA ligase from invitrogen. Then i directly use 1 ul for transformation into 30 ul of electrocompetent Top10 cells by electroporation.
The problem is that I got a lot of colonies in my negative control plate, although less than my other plates.
I know a re-ligation probably occured but does anyone know of anyway to verify the problem. I can only verify digestion for BamHI but not EcoRI since the latter digestion is on a linear plasmid and the cut out fragment is too small. Now I don't know whether to do colony PCR on the colonies since there is so much colonies in the negative control.
Posted 10 March 2009 - 07:50 PM
Posted 12 March 2009 - 02:57 AM
Just do a sequential digestion and CIP the vector, even if you have single cutters, they won't religate.
Posted 24 March 2009 - 11:51 PM
Posted 25 March 2009 - 12:16 AM
Its the other way round....shorter ones are easier to clone. The clones come in one shot and all the colonies that you screen are clones. (Well...if u follow the protocol properly
Thanks a lot. Although I have no time to do these because my deadline is coming near, I will definitely try these methods the next time. Anyway, is a shorter insert (~60 bp) more difficult to work with then a longer insert (~1 kb)....just curious...
Posted 25 March 2009 - 02:43 AM