Screning of a plasmid cDNA library

M.W. SMITH BMB6MWS at leeds.ac.uk
Thu Apr 11 10:10:49 EST 1996


Dear Yves,

Yes, you can screen your library with probes!
All of the protocols are in Maniatis and in my experience work well.
Firstly, you need to plate a diluted aliquot of your library onto a large 
agar plate (about 15-20cm square). You need a cell density such that you 
can pick off single colonies after the screen. Take a lift from this 
plate with a nitrocellulose filter (Sartorius sell reinforced 
nitrocellulose which is good for this use). Keep your master plate!
The bacteria on the filter are lysed, allowing the released DNA to bind 
to the nitrocellulose - protocol is in Maniatis vol 1 (?). Bake the 
filter at 80 degrees C and it is then ready for probing. I use 
random-primed 32P labelled probes. Hybridise and wash as stated in 
Maniatis. After autoradiography, you can align the film with the master 
plate to identify positives. To align these easily, I cut notches in the 
filters and draw the positions of these on the master plate. I usually 
pick several bacteria from the area of a positive, inoculate these onto a 
 gridded 9 by 9cm plate and repeat the screen. To ensure I have single 
colonies, I then streak the positives for single colonies, grid these out 
and screen one more time.
Good luck and I hope you get those clones!!
Mark Smith
Leeds University



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