Difference between revisions of "User:Chris D Hirst/Protocols"

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This protocol is desgined for use with the stratagene ''PfuUltra'' II Fusion DNA polymerase and is according to the DNA polymerase usage manual. [http://www.stratagene.com/manuals/600670.pdf| ''PfuUltra'' II Fusion Manual]

Revision as of 04:25, 6 August 2008


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


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.


Spectrophotometer Cuvettes P20, P200 and P1000 Gilsons 50mL flask (an additional one for each repeat) Timer


LB Medium LB Agar plates (x )


1. Grow up a culture of B.subtilis overnight

2. Pipette 500μL


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


Reagent Volume
Distilled H2O 16μL
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.

Protocol 3