Final desired concentrations are 20 mM HEPES, 2.5 mM Mg, 0.4 mM spermidine, 140 mm KAc, and 2 mM DTT.
WG is in buffer of 40 mM HEPES, 100 mM K, 5 mM Mg, 4 mM DTT.
Contributions from transcription using 10% of translation volume as transcript are 8 mM HEPES (pH 7.5), 1.4 mM Mg, 0.2 mM spd, 1 mM DTT. (Note: If you need to increase the % of transcript, you will have to consider the high Mg-concentration (14 mM) in the transcription reaction.)
For optimizing WG amounts from 10% to 40%, a different CB10X must be made for each reaction:
WG CB10X unlinked (volumes in uL)
10%
15%
20%
25%
30%
35%
40%
1M Hepes pH 7.6
160
140
120
100
80
60
40
4M KOAc
325
312
300
287
275
262
250
1M MgOAc2
20
17.5
15
12.5
10
7.5
5
0.1M spermidine
40
40
40
40
40
40
40
1M DTT
16
14
12
10
8
6
4
0.5M EDTA
2
2
2
2
2
2
2
Water
437
474
511
548
585
622
659
TOTAL
1
mL
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
WG CB10X 10% linked (volumes in uL)
10%
15%
20%
25%
1M Hepes pH 7.6
80
60
40
20
4M KOAc
325
312
300
287
1M MgOAc2
5
2.5
-
-
0.1M spermidine
20
20
20
20
1M DTT
6
4
2
-
0.5M EDTA
2
2
2
2
Water
562
599
636
670
Total
1 mL
1 mL
1 mL
1 mL
B. E mix - same as for RRL.C. WG - desalted, nuclease-treated. Current batches (Nov.'93) are typically used at 20% final concentration.
