B-gal assay

Howard Davis hdavis at mit.edu
Wed Aug 25 10:17:37 EST 1999


Hi Mimi,
  If you're looking for a homemade solution, here's one for you. We
regularly lyse NIH-3T3s to run an ONPG-based b-gal assay following
retrovirus transduction. The buffers & solutions we use are as follows:

Wash Buffer: 1mM MgCl2 in PBS
Lysis Buffer: 1mM MgCl2, 0.5% NP-40 in PBS
Substrate Buffer: 1mM MgCl2, 0.5% NP-40, 0.2%(w/v) ONPG in PBS
Stop Solution: 1M Na2CO3 in dH20

The procedure for a 96-well plate is:

-Wash cells with 100ul of wash buffer & discard
-Add 50 ul of lysis buffer and incubate for 30 min. @ RT
-Add 50 ul of substrate buffer (warmed to 37C) and incubate for 15 min. @
RT
-Add 20 ul of stop solution and incubate for 15 min. @ RT
-Measure OD420

The original reference for the application of this assay to retrovirus
gene transfer is:
Morgan JR, et al.           [See Related Articles]
     Rapid quantitation of recombinant retroviruses.
     Biotechnol Prog. 1994 Jul-Aug;10(4):441-6.

Please feel free to drop me an e-mail if you need any additional info.
Hope this helps!
   -Howard
Mimi Liao wrote:

> Hi all,
>
> Does anyone have a recipe for lysing mammalian cells for B-gal assay ?
>
> Thanks in advance
>
> Please reply to m.liao at student.unsw.edu.au




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