HELP re: probing phage libraries/hybridisation/+ve controls

Richard Hastings mbxrh at unicorn.ccc.nottingham.ac.uk
Mon Jul 31 09:35:13 EST 1995


I'm currently screening a cDNA library in lambda ZapII vector.  I'd
like a positive control to check my probe is hybridising well and to
check I'm not washing too stringently.  I'm using a 200bp partial PCR'd
sequence as a probe to obtain full length cDNA's.  What I thought of
doing was to dot blot some plasmid containing my 200bp sequence onto a
nylon membrane then hybridise and wash it alongside my plaque
transfers.  What I'd like to know is how much plasmid I need to dot
blot, ie I want to blot a similar quantity of DNA as would be taken
onto a membrane from a single plaque.  Am I asking a near impossible
question?  Any suggestions at all would be greatly appreciated.
Thanks again.

Richard Hastings (mbxrh at unicorn.nott.ac.uk)
Dept. of Biochemistry,
Queens Medical Centre,
University of Nottingham,
Nottingham NG7 2UH.



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