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			> Lysate Preparation of S-30
			> Membrane-Free Ribosomes (mfrs)
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			> Buffers and Reagents

 

Buffers and Reagents

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Part A: For S-30 prep

Growth media (4L)

22.4g KH2PO4

115.6g K2HPO4

40g yeast extract

Divide into 3 1L flasks, 5-100ml flasks, and 5-100ml bottles, autoclave. Prior to inoculation, add 40ml of 25% filtered glucose per litre of media.

Wash buffer (1L)

10mM Tris-acetate, pH 8.0

14mM Mg(acetate)2

60mM KCl

6mM B-mercaptoethanol (just prior to use)

50(g/mL PMSF (just prior to use)

Lysis buffer (100ml)

10mM Tris-acetate, pH 8.0

14mM Mg(acetate)2

60mM KCl

50(g/mL PMSF (just prior to use)

Preincubation mix (10ml)

3.75ml of 2M Tris-Ac, ph 8.0 (make up fresh)

213uL of 1M Mg (acetate)2

750uL of 19 aa's - met (1mM each)

37.5uL of 20mM methionine

600uL of 100mM ATP

200mg of phosphoenol pyruvate (PEP)

50 units pyruvate kinase

75uL of 1M DTT

H2O to 10 mL

Dialysis buffer (8L)

10mM Tris-Acetate, pH 8.0

14mM Mg(acetate)2

60mM K(acetate)

1mM DTT (just prior to use)

 

Part B: for mfrs prep:

TB (2L)

24g bactotryptone

48g yeast extract

8ml glycerol

Autoclave, allow to cool to < 60C, then add 200ml of sterile, filtered 0.17M KH2PO4, 0.72M K2HPO4.

Buffer A (100ml)

10mM Tris -Acetate, pH 8.0

14mM Mg (acetate)2

60mM K (acetate)

0.1mM DTT (just prior to use)

0.5mM PMSF (just prior to use)

Buffer B (200 mL)

10mM Tris-Acetate, pH 8.0

14mM Mg (acetate)2

60mM K (acetate)

0.5% Triton X - 100

0.1mM DTT (just prior to use)

Buffer C (100ml)

10mM Tris- Acetate, pH 8.0

14mM Mg (acetate)2

60mM K (acetate)

0.1mM DTT (just prior to use)

Part C: 2.5 X reaction mixture:

FINAL	STOCK	VOLUME/4mL of 2.5X (uL)
35mM Tris-Acetate pH 8.0	2M	175
190mM K(glutamate)	2M	950
30mM NH4OAc	5M	60
2mM DTT	1M	20
12mM Mg(OAc)2	1M	120
40uM 19aa's- met	1M	400
2mM ATP	100mM	200
0.5mM each of CTP, UTP, GTP	15mM	330
20mM phosphoenol pyruvate	400mM	500
0.1mg/mL E.coli tRNA	10mg/mL	100
35mg/mL PEG 8000	400mg/mL	875
20 ug/mL folinic acid	4000ug/mL	50
2mM IPTG	100mM	200
H2O to 4mL