Selection with puromycin

Victor Levenson levenson at uic.edu
Fri Aug 30 13:47:06 EST 1996


aiyar at ebv.oncology.wisc.edu (Ashok Aiyar) wrote:

>On 30 Aug 1996 13:08:37 GMT, Danny Altschuler <dla at galactose.mc.duke.edu> wrote:
>>Hey, I need to do a selection with Puromycin. Vector (pBabePuro - 
>>infection), cells HT29 colon carcinoma.). I know that I have to do a 
>>curve but could anyone give a good concentration range?
>>I have experience with Neo and Hygro and in my hands a good concentr. for 
>>these drugs is ~100-200ug/ml (hygro), and 400-600ug/ml active comp. (neo).
>>Which will be the magic number for puro??

>I posted a an almost identical question about two weeks ago and
>received no responses .....

>Going through the literature I have seen people use puromycin in
>the 2 - 5 ug/ml range for adherent cell lines, and 0.5 - 2 ug/ml
>for non-adherent cell lines.

>Ashok
>-- 
>Ashok Aiyar, Ph.D.                      
>Department of Oncology            email: aiyar at ebv.oncology.wisc.edu
>University of Wisconsin-Madison                  tel: (608) 262-6697

Hi, Danny and Ashok:


I am using puromycin selection on HT1080 cells (human fibrosarcoma)
and the "100% kill" concentration is 0.5- 1 ug/ml.  Puromycin is quick
and "clean"(no background) drug and in general I like using it. Some
cautionary notes, though:
1. My stock concentrations are in water at 1mg/ml, stored @ -20^C.
Freezing and thawing (3-5 times) seem to destroy the drug rapidly, so
you will be better off aliquotting it into "disposable" aliquots.
2. Apparently, puromycin looses its activity even @-20^C with time.
Recently we could not kill non-transduced cells even at 5 ug/ml and,
although I cannot be positive about it, 2 month storage is the limit.

Hope that helps,

Victor 




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