- October 23, 2012 at 6:33 pm #16958siddharthsameerParticipant
I am a master student and I am doing my master thesis in the cloning of keratinase sequence in competent cell.I tried several different methods and procedure for the cloning and I got the colonies but when I did the mini prep I had the good concentration of the DNA but when run on agarosei could not see the band.. I have to stop my thesis at this point but I am unable to think What could went wrong for the failure of the cloning, I am really confused what reasons should I put it in my discussion and in the overview that what should be done in future to avoid this proble.. Any help and answer would be of great help to me… thanks and regards
- October 28, 2012 at 1:28 am #112787CatParticipant
Increase antibiotic concentration in the culture you are using for mini-prep and for transformation. Most likely you have either false positives or your bacteria loose plasmids later.
- October 28, 2012 at 8:20 am #112791AstraSequiParticipant
There’s a lot more that can go wrong than just that. The exact possibilities would depend on what precisely you’re doing and which stage was the last one that you confirmed to be successful.
- November 14, 2012 at 1:44 pm #112974citroenboomParticipant
Some more data would be helpfull.
What kit did you use to get the DNA? How much did you load on the gel? Did you see the marker? Did you see other bands in the gel?
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