problems with DNA slot blots

John Hines hines at pharm.med.upenn.edu
Thu Feb 7 16:35:40 EST 2002


Hi everyone! Please help!
I have a problem that happens approximately every other time I make a DNA
blot to be tested with RNA probe. I use a number of cDNAs, all in similar
plasmids, among which there is a subset with shorter DNA inserts. This
"shorter" subset fails to bind RNA probe in every experiment, i. e., one
blot shows a perfect binding for all cDNAs, and in the other one made two
days later the bands corresponding to these "shorter" cDNAs are either
missing or very weak. Sometimes, the bands for one or two of these
problematic cDNAs do show up, while others are missing. So it is very
inconsistent. The other DNAs do bind in every experiment.  
DNA samples are prepared for blotting in a regular way: linearization,
dilution, denaturation with NaOH/EDTA and heating. RNA probe is OK.
Does anyone have an idea what can go wrong here?

Thank you 

Tania
tatiana5 at mail.med.upenn.edu




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