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
Distilled H2O	40.5μL
10Χ PfuUltra®II reaction buffer	5μL
dNTP mix (25mM each dNTP)	0.5μL
B.subtilis genomic DNA (100ng/μL)	1μL
Forward Primer (10μM)	1μL
Reverse Primer (10μM)	1μL
PfuUltra® II fusion HS DNA polymerase	1μL
Total Reaction Volume	50μL

Note. Template DNA should be diluted to 100ng/μL. If template DNA concentration is below 100ng/μL, 100ng of DNA should be added and the volume of H₂O to be added should adjusted to maintain a reaction volume of 50μL

Protocol

Add all the reagents in order (down the list) sequentially to the PCR tube mxing after each addition. Place PCR tubes into th ePCR machine and set the programme to the following set-up:

Initial Denaturation: 2 minutes at 95°C

30 Cycles of: 20 second denaturation at 95°C

                        20 second annealing time at Primer Tm - 5°C

                        15 second extending time at 72°C

Final Extension: 3 minutes at 72°C

The resulting solution can then be purified or gel assayed