Re: pcr

From: Alice L. Givan (Alice.L.Givan@dartmouth.edu)
Date: Thu Aug 23 2001 - 11:38:32 EST


Flowers,
I received several helpful replies to my question about methods for flow sorting single
cells into wells for pcr amplification.

Although there are many good articles,  the best general methodological reference that
I've seen is:

AB Kantor, CE Merrill, and JL Hillson (1996) Construction of cDNA from single
unstimulated mouse B lymphocytes: Method and application to the study of expressed
antibody repertoires in FACS-sorted murine B cell subsets.  Chapter 13 in Weir's
Handbook of Experimental Immunology (ed LA Herzenberg), Blackwell Science, Cambridge, MA.
pp 13.1 - 13.6

A tip mentioned in this reference,  and also reinforced in an e-mail to me from Larry
Arnold, is to adjust the voltage to the deflection plates in order to angle the waste
stream off to the side and aim the sort stream directly down into  pcr tube or well.
This increases the likelihood of the sorted cell arriving in the small volume of pcr
lysis buffer (and not getting stuck on the side of the tube).

Thanks for all your help.

Alice

Alice L. Givan
Englert Cell Analysis Laboratory
of the Norris Cotton Cancer Center
Dartmouth Medical School
Lebanon, New Hampshire NH 03756
tel 603-650-7661
fax 603-650-6130
givan@dartmouth.edu



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