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.
Preparation of XL1-Blue Electrocompetent cells
Prepaeration of E.coli cells for cloning of Biobricks and construct construction
Sterile Centrifugation bottles
1 Litre of LB medium (and appropriate antibiotics)
1-2 Litres of autoclaved and chilled ddH2O
10% glycerol in ddH2O, autoclaved and chilled
Dry ice bath
- Keep Everything Cold where possible
- Set aside an afternoon for this, starting the culture in the mroning
- Check the culture while growing frequently
- Grow up a culture of E.coli XL1-blue cells overnight
- Add 20mL of overnight culture to 1 Litre of LB medium (containing appropriate antibiotic)
- Grow cells while mixing at at least 225rpm until the culture reaches an OD600nm of 0.5-0.6 (1.6-1.9×108cells/mL)
- Test OD immediately after innoculating the Litre flask.
- First doubling may take 1 hour but doublings after that should be very 20-30 mins, so check often!
- When OD is 0.5-0.6, transfer the culture to 2 sterile 500mL centrifugation bottles and cool on ice for a few minutes
- Pellet cells in a centrifuge at 4000×g for 15 mins
- Quickly but carefully pour off the supernatant then carefully resuspend the cells in 10mL of ice cold ddH2O
- Fill both tubes to about 350mL with ice cold ddH2O
- Make sure the pellet is fully resuspended!
- Repellet the cells (as before) and again discard the supernatant
- Resuspend cells again in 10mL of ddH2O, then fill both tubes up to about 150mL with ice cold ddH2O
- Repellet the cells (as before)
- While repelleting, fill teh dry ice bath and set up eppendorf tubes (approximately 50) in a rack in the dry ice bath