IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 3.2: Difference between revisions
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==Steps== | ==Steps== | ||
#Fill an eppendorf tube with two samples (56ul x 2) of home made cell extract including reaction buffer, pyruvate kinase and rNTPs. | |||
#Fill a second eppendorf tube with two samples (40ul x 2) of commercial cell extract including the amino acids mixture, and preincubation buffer. | |||
#Place a quarter of each sample in a well in the 96 well plate, making up four wells. | |||
#In three of the wells, add 20ul of the DNA mixture. | |||
#In te last well, add nuclease free water (as a negative control). | |||
#Measure the fluorescence at 30 min intervals for the 1st hour, and hourly intervals thereafter. | |||
#After each measurement, cover the plate with the sticky lid. | |||
Revision as of 16:57, 28 August 2007
Equipments
- Fluorometer + PC
- 1 well plate
- Plate sticker lid
Reagents
- Pyruvate kinase
- rNTPs
- S30 cell extract (home made)
- Reaction buffer (home made)
- Commercial S30 cell extract
- Commercial Pre-incubation mix
- Amino Acids
- Minus Cysteine
- Minus Leucine
- pTet DNA plasmid
Steps
- Fill an eppendorf tube with two samples (56ul x 2) of home made cell extract including reaction buffer, pyruvate kinase and rNTPs.
- Fill a second eppendorf tube with two samples (40ul x 2) of commercial cell extract including the amino acids mixture, and preincubation buffer.
- Place a quarter of each sample in a well in the 96 well plate, making up four wells.
- In three of the wells, add 20ul of the DNA mixture.
- In te last well, add nuclease free water (as a negative control).
- Measure the fluorescence at 30 min intervals for the 1st hour, and hourly intervals thereafter.
- After each measurement, cover the plate with the sticky lid.
