At 04:58 PM 12/9/96 GMT, Tom Dougherty wrote:
>In article <32AB87C9.131A4030 at ix.netcom.com>, Robert Presswood <rpressw at ix.netcom.com> says:
>>>>J. Dennis Pollack wrote:
>>>>>> Help! I need a lysis technique to release cytoplasmic enzymes from transformed
>>> Ecoli. I cant denature the proteins and hence cant use some of the more
>>> frequently advertised techs. I suspect lysozyme will work, but I dont want to
>>> sonicate if I can avoid it. The procedures in Maniatis or Current Protocols
>>> are not detailed enough. Can anyone help? Thanks a lot. Dennis
>>The best method may be the alkaline Tris-Cl, EDTA & lysozyme method. This is
>similar to the DNA/plasmid extraction protocols, but if you omit the high glucose
>concentrations, the cells will readily lyse after a brief time at 37C. Sonication
>should not be necessary to obtain good lysis in this case. You may need to add
>excess MgSO4 and DNAse subsequent to lysis to decrease viscosity.
>>Tom Dougherty
>>
Do you then have to neutralize the alkali once the cells have lysed? How does the alkali affect the enzymes in the lysate in the first place?
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu
