User:Chris D Hirst/Protocols
Here will be placed any finished or currently worked on protocols until they are deemed of a high enough standard to be put on the Imperial iGEM 08 wetware wiki.
Cell Count v Optical Density Curve Calibration
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.
P20, P200 and P1000 Gilsons
50mL flask (an additional one for each repeat)
LB Medium LB Agar plates (x )
1. Grow up a culture of B.subtilis overnight
2. Prepare a 50mL flask of LB medium, adding the required antibiotics and take 1mL to use as a blank
2. Pipette 500μL into the 50mL of LB medium and mix thoroughly
3. Immediately take 1mL of the new culture and measure the OD against the blank
This protocol is desgined for use with the stratagene PfuUltra II Fusion DNA polymerase and is according to the DNA polymerase usage manual. PfuUltra II Fusion Manual
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).
Heated lid PCR machine
Thin walled PCR tube
|10Χ PfuUltra®II reaction buffer||5μL|
|dNTP mix (12.5mM each dNTP)||0.5μL|
|B.subtilis genomic DNA (50ng/μL)||1μL|
|Forward Primer (5μM)||1μL|
|Reverse Primer (5μM)||1μL|
|PfuUltra® II fusion HS DNA polymerase||0.5μL|
|Total Reaction Volume||25μL|
Note. Template DNA should be diluted to 100ng/μL. If template DNA concentration is below 100ng/μL, 50ng of DNA should be added and the volume of H2O to be added should adjusted to maintain a reaction volume of 25μL
The forward and reverse primers should contain the Biobrick prefix (forward primer) and the complementary sequence to the Biobrick suffix (reverse primer) 5' of the beginning of the annealing sequence
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 using a PCR purification column or by gel electrophoresis followed by spin purification.