Ligation gel picture interpretation
Posted 01 February 2012 - 09:05 AM
Control: Vector + ligase - - - - This gave approximately 50 colonies
1:3 Vector:Insert - - - - - - again around 50 colonies
1:8 Vector:Insert - - - - - around 60 colonies
I am worried about all of the self ligation that occurred and I doubt that any of my colonies on experimental plates are the clone I am looking for.
I ran some of the leftover ligation on a gel and would like some input on the multiple bands I observe in the vector + insert ligation reactions. I will be doing minipreps and XbaI/EcoRI digestions tomorrow so I will find out then if it worked for sure, but is the expected 7.5 kb vector+insert band in the smear?
lane 1: marker (12000, 8000, 6000, 5000, 4000...)
lane 2: control (vector + ligase)
lane 3: 1:3 vector:insert
lane 4: 1:8 vector:insert
Also, if you have any suggestions to limit self-ligation since my control plate had as many colonies as the actual ligation they would be appreciated. I have not used SAP or the likes for the vector since I have incompatible ends.
Thanks in advance
Posted 01 February 2012 - 09:33 AM
Posted 01 February 2012 - 09:42 AM
Posted 01 February 2012 - 10:24 AM
Posted 06 February 2012 - 12:08 PM