Posted 15 March 2013 - 12:53 PM
I am a fairly new grad student so I'm still learning a ton of tricks and getting a plethora of new projects to work on. My latest involves screening mutants that were created by a former grad student. This library was created using the mariner transposon. My P.I. suggested that I do rescue cloning to try and identify the genes that were disrupted. I tried looking up how to do this online and haven't really come across any protocols on how to do it. He gave me the basics but I would feel more comfortable if I got more specific details about it. Can anybody help out?
Posted 16 March 2013 - 02:04 PM
Easier, in my opinion, is to use inverse PCR. Design primers facing outward from your transposon. Cut and religate as above (it's ok if the enzyme cuts within the two primers). PCR using those primers, and amplify the religated fragment. Sequence. This will show the ends of the transposon and the region of the genome on each side, up to the RE cut site.
Another approach is to directly sequence genomic DNA. This works surprisingly well with modern sequencers and a good primer, for genomes at least up to 4 mb or so (E. coli size).
Posted 17 March 2013 - 03:19 PM