Southern Membranes from cosmids?

Hiranya S. Roychowdhury hroychow at nmsu.edu
Thu Apr 20 12:24:58 EST 2000


Hello,
        I deleted the original message from "Feng" earlier, so I am posting
this to the news group. I used to get similar low signal:noise ratio with
the cosmid library. The following method worked beautifully for me.

Cosmid Library Screening:

1.  Replica-plate the cosmid clones on rectangular pieces of HybondN. If the
library came in 96-well plates, 
using one of those 48- or 96-prong inoculator/replicator works beautifully.
2. Place the membranes, inoculated side up, on pyrex trays containing solid
LB-agar medium (w/ the 
appropriate antibiotic(s)). Incubate O/N at 37oC
3. Lyse the colonies in situ:
Place the membranes, colony-side up, on 2 layers of blotting paper soaked in
(1). 10% SDS ,  in  (2). the  denaturing solution ,  in (3). neutralizing
soln. and finally on blotting paper soaked in 20x SSC. Each treatment  is
carried out for 5min. 
4. Dab the membranes on paper towels and fix the NA by exposing to UV for
5-10min. (although 80oC vacuum  oven also works for fixing NA's onto nylon,
I would not do it in this case). 
5. After step 4, the colony blots are washed in 5xSSC, 0.5% SDS in a plastic
container jast barely large enough to fit the membranes flat. Put 5-6
filters in one dish and cover them with the SSC/SDS solution. Let them shake
vigorously (250-300rpm) on a table-top shaking platform. The jazzy rocking
platform will not work.
Keep shaking them with several changes of the washing solution every 30 min
or so till you do not see any cell  debris coming loose from the membranes.
Needless to say that you'll have to rearrange the membranes 
occassionally.
There have been occassions when I have had to do this for several hours, and
I'd leave it shaking at the last wash O/N. It is a small price to pay for
clean colony blots. 
I have also done the wash step before fixing the NA by UV, and the results
were not very much different in  terms of the signals. I thought that  I got
cleaner blots by doing it immediately after lysis. But if you are careful
not to let the blots dry off before you get to the washing step, it should
be OK.


Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu

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