@@ -225,31 +225,20 @@ The preparation of the cell extract used in a standard CFPS reaction.
#### Equipment
- Spectrophotometer
- Sonicator
- Ice water bath
- Centrifuge
#### Procedure
1. Inoculate a single colony of Vmax into 2-500mL of LBv2
2. Incubate at 30˚C overnight
3. After incubation, dilute 1:100 of culture into BHIv2 or modified 2xYT
4. Grow at 37˚C
5. T7 RNA polymerase expression is induced at OD600 = 0.6-0.8
6. Pellet cells once early stationary phase is reached (OD600 \>= 7.5) and wash three times in cold S30 Buffer
7. Resuspend pellets in 0.8 ml S30 per gram of wet cell mass and sonicate (20 kHz, 50% amplitude, 3x(45s ON/ 59s OFF), \~270 J/cycle, in ice-water bath)
8. Clear lystate at 12,000 g, 10 min, 4 C
9. Use supernatant for CFPS
### STANDARD CFPS REACTION ASSEMBLY
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@@ -261,7 +250,6 @@ plates depending on the scale of the reaction.
#### Equipment
- 1.5mL eppendorfs **OR**
- 96 well plate
If using 1.5 mL eppendorfs, the total volume per reaction is 30 uL\
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@@ -276,11 +264,8 @@ For 1.5 mL eppendorf tubes
#### Procedure for 96 well plate
1. Mix 1.5 mL of 2x Standard Buffer (-PB) with 800 ul of cleared VN cell extract.
2. Aliquot 23 ul of mixture to each of 96 wells.
3. Add designated additives to each well, then 400 ng of plasmid, then dH2O if necessary to final volume of 30 ul.
