Help with PCR

Benny Shomer bshomer at ebi.ac.uk
Fri Dec 29 10:28:47 EST 1995


avirup bose (abose at unlinfo.unl.edu) wrote:
: Hi Folks,
: I am planning to detect whether a recombinant vaccinia virus enters an
: animal cell. Assuming that the virus is unable to replicate, the 
: amount of viral DNA that will be inside the cell is beyond the limit 
: of detection of Southern Blot. So I am planning to do a PCR with the 
: isolated DNA to amplify the viral gene of interest and then do a 
: southern blot to confirm it's identity. Now my question: Does anybody 
: know of a protocol where you can do a PCR by directly picking up a few
: cells and using it as the template? I know we can do it with E.Coli 
: but I am not sure whether anyone has done that with animal cells. I 
: would appreciate it very much if someone can help me with this. Thanks
: in advance. My e-mail address is abose at unlinfo.unl.edu


Transfer the cells (from a few thousands up to 1-2 X 10^6) to an Eppendorf
tube containing 1ml of DDW, with 5-10% of Chelex-100 (BioRad, Technical
grade) and boil for 15-20 minutes. Let the tube cool and the chelex to
settle down and transfer 5-10ul of the supernatant to a PCR reaction
directly (depending on the reaction volume).  This works very well even
with up to 5ul of whole blood!!!  No disruption of the cells is needed.

You may have luck without the Chelex, but with the Chelex your'e on the
safe side.

Good luck,

Benny.
--
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Doing PCR?
Have you got some good, working primers???
Visit the PCR primers database at http://www.ebi.ac.uk/primers_home.html
and make your donation.

If you don't have access to WWW, just write to primers at ebi.ac.uk
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Benny Shomer
External Biological Liaison Officer,
EMBL outstation - The EBI,
Hinxton Hall, Hinxton,Cambridge CB10 1RQ, UK

Tel:   +44-223-494437
Fax:   +44-223-494468
Email: bshomer at EBI.ac.uk
http://www.ebi.ac.uk/ebi_docs/staff/benny.html

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~




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