ladasky@codon3.berkeley.edu (John Ladasky;1021 Solano No. 2;528-8666) (10/03/89)
Hi, remember me? I'm the "lowly undergraduate" from last year's
debate regarding publication of scientific results, peer review, etc. I have
run into a bit of a problem which may be familiar to some of you from your
grad-school experiences... a combination of time pressure and resource limi-
tations.
You see, I'm planning experiments which require the preparation of
tritiated thymidine-labeled SV40. I spent last semester doing cell culture,
and I intended to do experiments this semester. Only - oops - it seems that
something (like an enzyme) slipped past my phenol extraction, and now all of
my DNA is nicked. And hey, wouldn't you know it, I'm graduating in December.
I suppose that I could rewind the DNA with a topoisomerase, and
ligate it back together... but it's important that I have DNA that has a
linking number in the physiological range. Even if this were feasible, I
imagine that learning about this and experimenting with it would take several
weeks. So, here's my idea; my grad-student supervisor says that she has not
seen any commercial source of tritiated thymidine-labeled SV40 - but does any
one else know otherwise? I would be most grateful for this information.
Please email to: ladasky@enzyme.berkeley.edu (internet).
T CROSS POLICE LINE DO NOT CROSS POLICE LINE DO NOT CROSS POLICE LINE DO NOT CR
_______________________________________________________________________________
"Do unto others as you would like - John J. Ladasky ("ii")
to do unto them. " Richard Bach (ladasky@enzyme.berkeley.edu)elliston@msdrl.UUCP (Keith Elliston) (10/03/89)
In article <1989Oct2.221028.17141@agate.berkeley.edu>, ladasky@codon3.berkeley.edu (John Ladasky;1021 Solano No. 2;528-8666) writes: > > You see, I'm planning experiments which require the preparation of > tritiated thymidine-labeled SV40. I spent last semester doing cell culture, > and I intended to do experiments this semester. Only - oops - it seems that > something (like an enzyme) slipped past my phenol extraction, and now all of > my DNA is nicked. And hey, wouldn't you know it, I'm graduating in December. > > I suppose that I could rewind the DNA with a topoisomerase, and > ligate it back together... but it's important that I have DNA that has a > linking number in the physiological range. Even if this were feasible, I > imagine that learning about this and experimenting with it would take several > weeks. So, here's my idea; my grad-student supervisor says that she has not > seen any commercial source of tritiated thymidine-labeled SV40 - but does any One thing you could try (assuming that you still have complete circles) is to ligate in the presence of ethidium bromide. Ethidium br. intercalates between the bases, and unwinds the DNA about 26 degrees per molecule. If you play with the concentration you use for ligation, you should be able to modify the number of turns (and the linkage number) present in your SV40 molecules. You can easily assay this using agarose gel electrophoresis. Just be sure to extract the EtBr before you run the DNA on a gel. The principle is simple... Ethidium incorporates into your relaxed DNA, you add ligase and ATP to seal all the nicks in the presence of the ethidium. When you extract out the ethidium, you add 26 degrees per molecule of EtBr incorporated prior to ligation. You end up with negatively supercoiled DNA when you are done. When you run these molecules on a gel, you will see that the more the molecules are supercoiled, the faster they run in the gel. This is a simple experiment... set it up with some varying concentrations of Ethidium and see what you get as a result. It should only take a couple of days. I hope this helps.... If you have any questions, or want some further advice on the subject, drop me a line..... Keith Elliston uunet!msdrl!elliston P.S. I posted this so that other folks can add their $0.02 as well.