molecular cloning failure

[siddharthsameer]

molecular cloning failure

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Post by siddharthsameer » Tue Oct 23, 2012 6:33 pm

Hello
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

[Cat]

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Post by Cat » Sun Oct 28, 2012 1:28 am

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.

Good luck.

[AstraSequi]

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Post by AstraSequi » Sun Oct 28, 2012 8:20 am

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.

[citroenboom]

Re: molecular cloning failure

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Post by citroenboom » Wed Nov 14, 2012 1:44 pm

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?