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
Harry.Witchel at bristol.ac.uk