oocyte RT - (Apr/05/2001 )
I want to screen a phage cDNA library with Digoxigenin-labeled probe. Can I use the same hybridization protocol as with a 32P-labeled probe or does specific protocol exist ?
I am doing something similar. However, my cDNA library is not a phage library it is in E. coli.Are you using a Kit? I have been using a kit from Roche. It works well enough. I suggest some modifications to the protocol they give. Such as every membrane be washed and probed and developed seperately. For colony lifts let the membrane dry, then UV cross link and then wash in 2xSSC before pre-hybridization and probing this reduces colony debree which is a source of background. If you want more info let me know.
Hi, you should order the free >DIG's system User Guide For Filter Hybridization< from Boehringer Mannheim. There are good protocols and tips. A friend of mine got very good results using this stuff.
Hello there!I already did some RT-reactions with Xenopus-oocyte RNA. This way I got Xenopus TBP (TATAbox binding protein) and UBF (upstream binding factor).