DNA FISH problems - Help needed! (Oct/16/2007 )
Has anyone tried in situ hybridization (ISH) using DNA probes and target DNA sequences (as opposed to RNA)? I have a protocol for RNA FISH optimized for the EUB338 probe that I'm adjusting to work for DNA sequences but I still haven't been able to get ANY fluorescence. My protocol is as follows:
PFA fixation of E. coli cells
Detergent- tween 20
Spot onto gelatin slides and heat
Denature DNA with 0.1N NaOH for 90s at 37 deg (then rinse off) or Denature DNA at 70 deg for 2 mins in 70% formamide
Dehydrate in alcohol dilutions (50-95%)
Add hybridization cocktail (formamide buffer + HEX-labelled probe) overnight in hyb oven
Wash off probe with wash buffer
Rinse and dry slides
I'm trying to detect virulence genes, which have a very low copy number so I was expecting to get very little fluorescence. But I wasn't expecting to get absolutely nothing with a pure culture of E. coli cells!
Has anyone tried DNA FISH before? What could I be doing wrong? Is it my protocol?
I master the FISH, but don't know your protocol never. It looks too simple to get any result.
Did you do some DNA FISH?
What are your DNA probed, DIG, Biotin or others?
thanks for the reply seablue,
simple, how so? is the Tween 20 too weak of a detergent here, or is my hybridization step the problem? Are there any tried and tested DNA ISH protocols you can suggest instead? My probes are labelled with fluorochromes (HEX and Cy5), and unfortunatley the few protocols I have come across online are for DIG probes, so I really had very little to go on when I tried to modify a colleague's RNA FISH protocol.
any suggestions are welcome- thanks!