[TAs
? 5 AAS

1/11/93

L

Attention: Vittorio Sgaramelia
Department of Microbiology and Genetics
University of Pavia

From: Greg Tombline
Rockefeller University, Lederberg lab

Dear Vittorio,

Since my last letter | have been trying to clarify the
fingerprinting on the mutants and have the sequence side by side for
both P3 and P4 ...... the last gel was beautifully resolved but
unfortunately ripped and folded right at the spot of the mutations.
The gel consisted of the new protocol which | mentioned to you
before, ie. using 8% Hydrolink with 40% formamide and normal (not
wedged) spacers. | believe that the samples were overheated the
last time | used this method - the lanes appeared blurry. So, in
order to avoid this | heat the samples for approx. 5-7 minutes, then
chill on ice approx. same time, then load. This last gel was really
nice, but just hard to handle. We still have more of the samples..
good sequencing with sequenase, and fingerprinting done with 32P
instead of 35S - was extracted and precipitated. So, | will do my
best to make the perfect gel and handle it most carefully. From
what | could see there were no gross/ indirect effects of the
mutations on the fingerprint for at least P3, but P4 might be
slightly more complicated. Don't lose hope, | really think that we
are almost there. |'m making another gel today with wedged spacers
but 8% with 40% formamide and will run it tomorrow (Tues
1/12/93), so we should have something Wed. ... this gel runs approx.
7 to 8 hours...... patience...

In the arena of endonuclease..... | haven't been able to do very
much since our glass plates have been limited and I've wanted to
knock off the fingerprinting.... Have you found anything new?

In David's arena things have been rough. | am trying to finish
controls . will let you know. We also have a new member- Sri
Sastri has joined , so we're trying to make space.

So for now, Happy New Year. Non Prevalabunt....
I'm back.... it's monday 1/25/93 now. The last three gels have
failed in the sense that they were all blurry, although counts were
incorporated. | have been experiencing real difficulty with handling
the gels in general.. they have been sticking to the plates. | suspect
that the formamide gels might be the root of the problem......but it
worked really well at least once. It has been very disappointing.

On friday we received some new glass plates, that is to say, we are
no longer limited by plates, so | will run two gels, one 6%

acrylamide without formamide, and another. with Hydrolink +
formamide. The reactions should still be good - they are only a few
days old. Meanwhile | will also take the autoradiograph of the nice
gel (Hydrolink + formamide; which ripped in the immediate are of the
mutations) to the media services and send you a copy- to show you
how nice this type of gel resolves (with no compressions of "G"
bands and very little smiling) so you can appreciate why | am trying
to use this technique. | am really sorry that it has been taking so
long, as | said it is really frustrating to put in so many hours for
nothing.

| am hoping that the controls for David's experiments will yield
some conclusive data. | have finally found a way to destroy the RNA
which was supposedly DNAsed after transcription (alkali then
dializing on a .O25micrometer millipore membrane) and have also
shown that DNA primers do not slip through-- then the synthesis is
not hindered- this is to show that the DNAse treatment after the
transcription is complete and that the supposed RNA generated
mutants are not the result of any DNA (of the PCR product which the
RNA was transcribed off) which might have survived. We ran out of
ssccc uracil containing phagescript (Template for the synthesis
reactions) - and generating new, clean, homogeneous stuff has been
time consuming as well- but | think that we have some now. As for
the RNA primer in a PCR, it's been on hold, but plan to start soon
again as well as making 30 and 60 base RNA to use in the
mutagenesis expts as well as RNA PCR.

Julie has started some good experiments involving ELISA in the
detection of RNA/DNA hybrids, as well as RecA protein with
antibodies, so far with nice looking controls.

Helene is back from France and starting expts which | believe
corolate in vivo and in vitro studies of formaldehyde mutagenesis
using’ phagescript as the substrate. | am sure that Dr. Lederberg will
keep you up to date with it.
David has been following up the analysis of his RecD mutants
which are highly resistant to ds breaks and |' sure has a ton of
data.... it should be very interesting.

Ken met with some success in purifying his heteroduplexes
which he made by PCR, and started transcribing off of them.... this
should also be interesting. Right now he's trying to resolve things
with Quin ling - he has started the divorce proceedings, and has
attempted to bring her back to China. We wish him luck, he has
seemed somewhat less stressed.

Mike has had some interesting ideas which he will probably tell
you about- involving organization of databases in relation to the
Human Genome Project and organizing molecular genetic info in
general. He is also working on gel analysis with Mic, and a possible
spreadsheet format for looking at a PCR reaction....

As | said, Srinivas Sastri has arrived and is settling in-- no
bench work yet...

We have had a couple of good seminars... Bruce Ames spoke and
also visited the lab. | also attended a seminar entitled " From the
crime scene to the courtroom, and the biology of forensic medicine"
-- mentioned were RFLP analsis which although out of date? is the
most commonly used technique by the FBI... fingerprinting by other
means was only briefly mentioned... Anyway, today Sir Walter Bodmer
will give the Joshua Lederberg lecture in molecular genetics on
human polyposis and muation rates in humans- should be a good one.

As for my love life, things are great. Robin sends her best.

So for the moment, stay well , will let you know what happens.

ve