Hello Dr. Perumal

I have few suggestions for your experiment on R-genes....with an assumption that your objective is to isolate all the genes (if this is correct kindly let me know) from the BACs..

1) If you have sequences available ...do an multiple alignment to see the region of differences ....whether UTR or coding region...

2) Then you PCR amplify specific gene specific regions and use them as probes

3) If mRNA/coding region isolation is your basic objective...do a reverse transcription using specific UTR primers and isolate full length genes in the second step

4) What kind of probes?..this query is not very clear to me kindly elaborate

5) DIG based detection is associated with soem background.....so maya have to optimize little bit ...it using for the first time....on colony or filter hybridizations .....it may problematic sommetimes........I'll suggest Radioactivity based detection..in place of DIG...it takes less time and is generally more clean and sensitive...

bye

Dear Ajay

Thanks for the response.

By 'kind of probe' I meant the length of the probe (template), radioactive versus fluorescent labeled, random primed versus overgo etc. etc. I would like to avoid radioactivity if possible, but if that is the best option, I do not want to waste time and resources running after the fluorescence option. The Roche DIG Filter hybridization protocol is what I am considering, but have my doubts about its applicability to probing BAC filters.

I welcom any suggestion that will help me decide.

Thanks