diff --git a/pages/usermanual.mdx b/pages/usermanual.mdx index 64493240149c81b255d733d4b4d1d7fad05d42c6..99205b3a312de65082cb788c5096b28750bd4531 100644 --- a/pages/usermanual.mdx +++ b/pages/usermanual.mdx @@ -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 30ËšC overnight - 3. After incubation, dilute 1:100 of culture into BHIv2 or modified 2xYT - 4. Grow at 37ËšC - 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 @@ -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\ @@ -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. - 4. Foam the reaction ## Safety