Harry.Witchel at bristol.ac.uk (Harry) wrote:
> 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
> Happy cloning!
>>Harry.Witchel at bristol.ac.uk>
1) use a set of primer directed against conserved domains of your gene and do PCR with all your clones (to get the info which one is the right one), if that doesn't work, do PCR with T3 and T7 primers. You get an idea of the insert size and you can decide whether it matches the expected size.
If you have several products of the same size, make a restriction analysis: cut with a (or several) restriction endonucleases and separate them on a gel: you get a good idea which clones are identical which can save you much sequencing work
2)Instead radioaktive ink (which gives usually pretty big spots) you can use phosphorescent ink. You can get those glow-in-the-dark inks in crafts supplies. Put one small spot of ink at the position where the holes are (onto the Saran wrap; I use thin sheet protectors instead of Saran film because its easier and faster to handle), let it air dry (!) and expose it to normal room light, then expose to the film. You can also put the film onto your filter (before or after exposure) and poke holes in the film where your holes in the filter are.
Hope that helps, Thomas