rescue cloning of a transposon mutant - (Mar/15/2013 )
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?
Some transposons carry a bacterial origin of replication, and if they are cut out and recircularized, they can transform E. coli cells. I don't know if your transposon does this. Even if not, then what you want to do is similar -- find a frequent cutting enzyme that cuts your organism's DNA, but is not present in your transposon. Often a 4 base cutter, such as Sau3AI is good. Cut with it and then religate. Transform E. coli. You can sequence the plasmid to determine the transposon insertion location.
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).
Thanks! That sounds a lot easier than what I had imagined. I'm going to try this with in the week to see how it goes. Thank you again!