I am screening the clones obtained after successfully transecting the targeting vector into ES cells. After neomycin selection, the cells were expanded and Now, I am planing for screening to detect positive clones. Does any alternative molecular methods except southern blot and PCR, which helps in screening positive clones?
You could try designing a probe and doing DNA FISH? That would let you see if you have multiple insertions too.
Extract the vector (it is not inserted into the chromosome), make a linear form with restriction enzyme and make sure it is your interested size by agarose gel analysis.
You could try designing a probe and doing DNA FISH? That would let you see if you have multiple insertions too.
@Jorg: From the question I infer that there are at least two genes (GOI + Neo) that have been inserted into the genome. Oligos + labelling kit and away it goes. Now, did you want to help answer Praveen's question or did you just want to aim a heavy dose of sarcasm at me?
From a practical point of view there are no alternative methods to screen positive clones. You should go to screen by PCR and then confirm by southern blot.
you can go for DNA macro-array.. you isolate the DNA from different clones (or yu can stick the clones itself) onto nylon membrane dry them and identify with radiolabeled probe. You can screen a really large no. of clones through this technique
Consider genomic qPCR... good reference here:
http://www.ncbi.nlm.nih.gov/pubmed/17332643
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods