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)
Lots of LB Agar plates
Be VERY careful to avoid contamination at all stages
- Grow up a culture of B.subtilis overnight
- Prepare a 50mL flask of LB medium, adding the required antibiotics (if any) and take 1mL to use as a blank
- Pipette 500μL into the 50mL of LB medium and mix thoroughly
- Immediately take 1mL of the new culture and measure the OD against the blank
- Dilute 100μL of culture in 900μL of LB and mix well
- Plate 100μL of this diluted solution
- Repeat the dilution and plating 8 times to obtain plates for colony counting
- Be sure to mark the OD, time and dilution for each plate!
- At 60 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes take further 1 mL samples, measure OD, dilute and plate
- The Following day, check all plates to determine which plate at each time point is the most crowded plate on which all teh colonies can still be counted individually.
- The cells on these plates should be counted, and the number of colonies mutiplied by the dilution factor to calculate the number of cells in 1mL
- Plot a calibration curve of OD against cells per mL
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 morning
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 the dry ice bath and set up eppendorf tubes (approximately 50) in a rack in the dry ice bath
- Pour off the supernatant and resuspend the cells in 20mL of 10% glycerol (resuspend one pellet then transfer the solution to the other bottle and resuspend the second pellet)
- Transfer the cells and glycerol solution to a sterile 50mL centrifuge tube and pellet for 15 mins at 4000×g
- Pour off supernatant and resuspend pellet in 2mL of 10% glycerol
- Pipette 50μL aliquots into the eppendorfs in the dry ice bath
- Feeze on dry ice
- Depending on pipetting accuracy, between 50 and 60 aliquots should be made
- using a repeating pipetter makes this proces much faster and reduces risk of contamination
- Store at -70°C
Biomaterial Secretion Testing by gel
To test for expression of biomaterials in B.subtilis and to determine if the biomaterials when expressed are being secreted or are remaining within the cell. This can also be extended to find optimal conditions for production and secretion.
Vertical Gel electrophoresis tank and equipment
Coomassie Blue dye
Destain soltuion (%methanol,%,%)
LB medium (and antibiotics)
Preparing a polyacrylamide gel
Production of Samples
- A 1mL sample of B.subtilis lacking the construct should be prepared by the following method and aliquots stored in PAGE buffer
- Grow a culture of B.subtilis in 5mL of LB medium with antibiotics overnight
- Take 50μL of overnight culture and inoculate 5mL of LB medium (with antibiotics) and grow until OD600 reaches 0.4-0.6
- When OD reaches 0.4-0.6, take a 1mL sample then induce expression using a set concentration of inducer
- Inducer is probably IPTG, xylose or light and each should have a specific concentration for use (determined previously)
- Grow at 37°C for a pre-set amount of time (probably 1 hour or 4 hours but may be more)
- When the culture has been allowed to grow for the set amount of time (or has reached a time point if using time points), harvest 1 mL of the culture and pipette into an eppendorf.
Preparation of Samples
- Centrifuge the sample for 5 minutes at 13000rpm
- Being careful not to disturb the pellet, pipette off the supernatant into a separate eppendorf
- Resuspend the pellet in μL of PBS
- Pipette μL of the pellet in PBS and the supernatant into separate tubes.
- Mix each sample with
Loading and running the Gel
- Boil all samples at °C for 5 minutes
- Carefully mix each sample before loading by rubbing tube between fingers and load 15μL of sample into the wells, taking care to note which sample is in which well on which gel (if 2 are being loaded simultaneously).
- When loading to gels, load along in one direction, alternating between the two gels (if both used), but beginning with the rear gel.
- Be careful to load sample into a single well and not contaminate adjacent wells and avoid bubbles where possible
- Gel should be run for approximately 70 minutes at 130V, or until the blue gel front has been eluted from the bottom of the gel
- Check for bubble rising to ensure the gel is running correctly