CELL FREE SYSTEMS	> TRANSLATION	> E coli Translation	> Translation Protocol
			> Sample Protocol
			> Troubleshooting
			> Lysate Preparation of S-30
			> Membrane-Free Ribosomes (mfrs)
			> Ribosomal Pellet
			> Dialysis Tubing Work-up
			> Membrane-Free Translation System
			> Buffers and Reagents

 

Membrane-Free Ribosomes (mfrs)

Get a printable version of this protocol (requires Adobe Acrobat)  The removal of endogenous vesicles also removes a large fraction of ribosomes, which must be added back to the reaction exogenously. The procedure for isolation of mfrs is as follows:

1. Inoculate 1.5L of TB + 0.2% glucose with 100ml saturated culture of MRE 600 cells. Grow to OD600 of 1.0 at 30ºC. Pellet cells (5000 rpm, 15 minutes), and wash 1X with ddH₂O. Resuspend in buffer A at 0.5g/ml Pass suspension through French pressure cell 3X at 4000psi Centrifuge 30 min. at 40 00xg (19 000 rpm in 50.2). Dilute supernated 10- fold in buffer B. Centrifuge 2 hours at 150 000xg (37 000 rpm in Ti 50.2). Resuspend pellet in 18 mL of buffer B (Note: use Dounce homogenizer with type A pestle). Incubate vernight with rotation at 4ºC. Centrifuge 2 hours at 150 000xg. Resuspend in 20 mL of buffer C. Centrifuge 2 hours at 150 000xg. Note: Pellet is almost clean, and has approximate consistency of "snot".
2. Resuspend pellet in 500ml buffer C.

   Note: Volume of mfrs required must be determined, but should be optimal in this case at about 0.1ml per 10ml reaction.