Composition of M9 media.

[John K. Troyer]

 On 11 Oct 1995 sl3wx at cc.usu.edu wrote
> 
 > Hi netters,
 > I would be grateful for some information regarding minimal medium. 
 > I am trying to express a protease via E.coli XL-1 . The bug grows very 
 > slowly in LB media (the expression vector is pIN-III-ompA 2 ) and I can 
 > see a band on a Cooomassie Blue gel at a position corresponding to the 
 > pure enzyme. However the expression level is not very high and I suspect
 > the promoter may be leaky. I tried M9 media (according to Maniatis) but 
 > after innoculation of a colony into 5ml media the flask shows no turbidity
 > even after 24 hrs. 
 > My questions are
 > 1) Can anyone provide me with a recipe for Minimal Media that worked fine
 > for them???
 > 2) How long is one expected to wait to observe growth??
 >  Look forward to assistance.
 > Thanks in advance
 > ____Ventris_____
 
 
 Ventris:
 
 I have had much better success plating out E coli on M-9 plates first, 
 and then picking a colony from the M-9 plate to inoculate into liquid 
 media.  The plating efficiency of E coli which have been grown on a rich 
 media is rather low when transferred to M-9.  Therefore, plating first is 
 almost essential.  The recipe I use for plates is as follows:
 
 6 g   Na2HPO4
 3 g   KH2PO4
 0.5 g NaCl
 1 g   NH4Cl
 15 g  agarose 
 qs to 1 L with water and autoclave.  When media is cooled to around 50 C. 
 add the following:
 
 2 ml 1M MgSO4
 0.1 ml 1M CaCl2
 10 ml 20% glucose
 1 ml 1M Thiamine-HCl
 
 Hope this helps.
 
 Regards,
 
 John
 
 ******************************************
 John K. Troyer, PhD
 University of Maryland School of Medicine
 Department of Biological Chemistry
 (401) 706-7518
 jtroyer at umabnet.ab.umd.edu
 ******************************************