Screning of a plasmid cDNA library
BMB6MWS at leeds.ac.uk
Thu Apr 11 10:10:49 EST 1996
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!!
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