User:Chris D Hirst/Protocols
Protocols
Here will be placed any finished or currently worked on protocols until tehy are deemed of a high enough standard to be put on the Imperial iGEM 08 wetware wiki.
Cell Count v Optical Density Curve Calibration
Aim
To produce a calibration curve to aid in the normalising of fluorescence values to allow proper characterisation of Promoters and RBSs for B.subitlis. This protocol must give results that are as accurate as possible over a considerable range of Optical Densities.
Equipment
Spectrophotometer Cuvettes P20, P200 and P1000 Gilsons 50mL flask (an additional one for each repeat) Timer
Reagents
LB Medium LB Agar plates (x )
Protocol
1. Grow up a culture of B.subtilis overnight 2. Pipette 500μL
PCR
Aim
To produce clones of two genes from B.subtilis that are too big to have synthesised by GeneArt; for use as integration sequences (AmyE and PyrT) or for their original purpose (XylR).
Equipment
A heated lid PCR machine A PCR tube
Reagents
| Reagent | Volume |
|---|---|
| 40.5μL | |
| 5μL | |
| 0.5μL | |
| 1μL | |
| 1μL | |
| 1μL | |
| 1μL | |
| 50μL |
Note. Template DNA should be diluted to 100ng/μL, if template DNA concentratino is below 100ng/μL however, a 100ng of DNA should be added and the amount of H2O to be adjusted to maintain a reaction volume of 50μL
