Single-copy eukaryotic Southern...
levenson at uic.edu
Thu Jun 5 13:55:53 EST 1997
mbthiam at NEXUS.MICROIMM.MCGILL.CA wrote:
>If anyone could help me out it would be much appreciated!
>I am looking for protocols that would allow me to do single-copy
>eukaryotic genomic Southerns, as I am looking for possible homologues
>to a human gene in other eukaryotic organisms (mouse, rat,
>chicken, Guinea pig, calf, ...). My problem lies in the fact that, using
>a 294 bp cDNA fragment which encodes the DNA-binding
>region (88 aa) of the much larger human protein (1093 aa), I have not
>been able to get a signal when performing a Southern with my
>control (human epithelial cell genomic DNA).
>Thanks for your time and attention!
>Madani B. Thiam
>Ph.D. student, Dept. of Microbiology and Immunology
>mbthiam at microimm.mcgill.ca
Insufficient data for a meaningful answer.
1. How much control DNA was loaded per lane? What RE was used and what
size fragment do you expect to detect?
2.Details of transfer protocol: acid apurinization, transfer buffer,
membrane used, weight (if classical upward transfer), time of the
transfer, treatment of the membrane afterwards.
3. Details of hybridization: efficiency of the labelling reaction,
amount of probe in the reaction, clean-up procedure, buffer (mol
crouders, SDS, salt), conc of probe in hybr, time and temp of hybr,
time and stringency of wash and theis ##.
4. Exposure conditions: time temp film, screen.
On a general note, you can detect a single copy gene if you load 6-10
ug of digested DNA per 7 mm lane, trasnfer to HybondN+ (no, I am
not...) after 30 min in 0.4 M HCl in 0.4 M NaOH o/n, and hybridize in
dextran-containing buffer o/n at 62^C in 5 ml using 20 ng of labelled
probe (efficiency approx 60%); exposure - using pre-flashed Kodak film
(no, I am not...) and screens at -70^C - for about 2-3 days.
Victor Levenson MD/PhD
Res Asst Prof
UIC, Dept of Genetics M/C 669
900 S Ashland Ave
Chicago IL 60607
levenson at uic.edu
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