Hello All
when we try to prepare 10 ml of this solution and shaking by hand the solution couldn't be soluble
lysis buffer containing 10 mmol/L Tris-HCl pH 7.5, 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5 mmol/L b-mercaptoethanol,0.5% (w/v) CHAPS (Pierce, Rockford, IL), and 10% (v/v) glycerol.
WHAT can we do ?? is it need heating --------Note: we annot prepare more than 10 ml as the ingredients we have are of low quantities.
PLEASE HELP
Beat regards
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2 replies to this topic
#2
phage434
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Posted 22 May 2010 - 06:23 PM
The EGTA will not go into solution without neutralization with sodium hydroxide. Prepare a 100 mM solution at pH 8.0 or so by adding sufficient NaOH.
The PMSF will not dissolve in water very well; use isopropanol instead to make a stock solution.
Dilute the two stock solutions to make the final lysis buffer. It will be easier to also start with stock solutions of Tris, CHAPS, and MgCl2 and make the lysis buffer by mixing solutions in the proper amounts rather than by weighing components and dissolving the resulting powder mixture.
The PMSF will not dissolve in water very well; use isopropanol instead to make a stock solution.
Dilute the two stock solutions to make the final lysis buffer. It will be easier to also start with stock solutions of Tris, CHAPS, and MgCl2 and make the lysis buffer by mixing solutions in the proper amounts rather than by weighing components and dissolving the resulting powder mixture.
#3
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Posted 23 May 2010 - 06:01 AM
jehane, on May 22 2010, 10:16 PM, said:
Hello All
when we try to prepare 10 ml of this solution and shaking by hand the solution couldn't be soluble
lysis buffer containing 10 mmol/L Tris-HCl pH 7.5, 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5 mmol/L b-mercaptoethanol,0.5% (w/v) CHAPS (Pierce, Rockford, IL), and 10% (v/v) glycerol.
WHAT can we do ?? is it need heating --------Note: we annot prepare more than 10 ml as the ingredients we have are of low quantities.
PLEASE HELP
Beat regards
when we try to prepare 10 ml of this solution and shaking by hand the solution couldn't be soluble
lysis buffer containing 10 mmol/L Tris-HCl pH 7.5, 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5 mmol/L b-mercaptoethanol,0.5% (w/v) CHAPS (Pierce, Rockford, IL), and 10% (v/v) glycerol.
WHAT can we do ?? is it need heating --------Note: we annot prepare more than 10 ml as the ingredients we have are of low quantities.
PLEASE HELP
Beat regards
use stock solutions of pre-sovled EGTA (solved in Tris), and PMSF (solved in isopropanol)
