Choosing clones from a phage library for analysis

Harry Harry.Witchel at bristol.ac.uk
Sun Nov 19 11:53:02 EST 1995


Hello Phage-Kings!
	I am just finishing my tertiary screen of a Zap II library, and it 
looks like I have a large number of clones that bind my random primed 
radioactive probe.  Ultimately my screening "assay" is sequencing the clones 
and judging whether the sequence matches what I (and the EMBL data bank) think 
is a K+ channel.
	My question is: what is a reasonable and time-efficient way of 
choosing from among the clones which bound the probe.  How many clones should 
I use from each tertiary plate?  What is the most time-efficient method of 
determining insert length starting from coring the phage out of the agarose?  
What is the best sequencing strategy for quickly figuring out whether a clone 
is the gene of interest?
	Also I am having trouble getting my allignment marks to be perfect 
when doing the autoradiographs of my plaque screens.  Currently I place the 
filters on a saran wrapped piece of stiff film, then cover the filters with 
another piece of saran wrap.  The filters have syringe needle holes punched in 
them, and a small amount of ink surrounds each needle hole.  I place small (1 
x 2 cm) sticky labels over the holes, then dot the labels (the ink spots on 
the filters are somewhat visible through the label) with radioactive ink.  
Once the ink dries, I cover the the sticky label with another sticky label (to 
prevent radioactivity from transfering directly onto the film), and then I put 
everything into film cassettes.  Radioactive ink is made by dosing a small pot 
of ink with very little 32P (1 ul, which still makes the ink screaming hot), 
and then when needed, a small amount of the hot ink is diluted down with 
normal ink, a felt tip marker is dipped into the ink, and after eliminating 
excess ink by making a practice spot, spots are dotted onto labels such that a 
label makes 2-5 counts when held in front of a minimonitor.  Labels with too 
much radioactivity can be eliminated before applying them to the filter set.
	Is there a sane way to make radioactive ink?  Is there a better or 
more consistent pen for radioactivity rather than just using an old marker?  
Is there an easier way of seeing EXACTLY  where the needle hole is (right now 
my ink spots have a 2 mm margin for error)?  I prefer spotting the allignment 
holes on the filters rather than spotting the back board and alligning later 
using the filter holes directly because it seems to me that one would get less 
slippage by missing out the step where the autorad is realligned with the 
backboard and then the needle holes from the filters are drawn onto the 
autorad.
	Happy cloning!
	Harry

Harry.Witchel at bristol.ac.uk




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