LiCl Cell wall wash
LiCl Cell wall wash
1- Pellet cells 4 min at 4.5K in F14-6x250y rotor.
2- Suspend in a total of 8 ml pH 7 MOPS buffer
3- Take 4x2 ml aliquots (screw cap conical tubes)
4- To A add 65 microliters 10M LiCl (0.3M), to B add 130 microliters 10M LiCl (0.6M), to C add 260 microliters 10M LiCl (1.2M), to D add 550 microliters 10M LiCl (2.4M)
5- Incubate 15 min at RT, then pellet cells (3min at 2K in S4180 rotor).
6- Save supernatants (cell wall wash), ppt 1ml aliquots of each w/TCA.
7- Dissolve pellets in 30 microliters borate cracking buffer w/2mM IAm.
8- 100C for 4 min, then run 4, 12 microliter aliquots on SDS gels