Team:British Columbia/Notebook

Notebook: Need to Know

Standard Operating Protocols (SOPS)

Colony PCR

Supplies Needed:

1. PCR tubes
2. BioBrick PCR primers (G1004, G1005) or (VF2, VR)
3. Taq Polymerase
4. 10x Reaction Buffer
5. 10mM dNTPs
6. sdH2O
7. Colonies to be PCR’ed
8. Agar plate for indexing

Steps:

1. Make master mix of primers and other PCR components EXCEPT Taq polymerase. Keep on ice.

PCR Master mix

Reagent	1x rxn volume (uL)		Master Mix
5x rxn buffer	5	xn
10mM dNTP	0.5	xn
sdH2O	9.15	xn
Phusion polymerase	0.1	xn
MgCl2	2	xn
DMSO - 5%	1.25	xn
gDNA	3	xn
Total	25

N = number of PCR tubes/samples

Make sure to add about 2 extra samples to account for pipetting error and 1 extra sample for water control.

Example: for 20 colonies, let N be: 20 (colonies) + 2 (extra)+ 1 (water) = 23

2. Add Taq polymerase.
3. Aliquot 25uL per PCR tube. Keep the PCR tubes on ice.
• *Following steps must be done near flame*
4. Touch toothpick/pipet tip/loop to colony, then index plate, then swirl around in PCR tube.
• *Turn flame off*
5. Run PCR:
• Turn machine on
• Load samples on the machine
• Select iGEM
• Select screen
• Make appropriate adjustments to the temperatures and times
1. 95°C for 15 mins
2. 94°C for 30 secs
3. 56°C for 30 secs
4. 68°C for 1 min per kb of expected product

*Round up for this step. i.e. For a 3.6kb construct, use 4 min elongation time.
5. Repeat 2-4 30 times.
6. 68°C for 20 mins
7. 10°C forever
8. Once finished, remove the PCR tubes from the machine.
6. Verify PCR products on agarose gel OR store the PCR tubes in the 4°C walk-in fridge until they can be verified on agarose gel.

Gel Verification (adapted from ‘Molecular Biology Laboratory 2010 iGEM Training Workshop’)

Supplies needed:
1. Agarose (1g/100mL for a 1% gel)
2. 1X TBE Buffer (dilute 10X TBE into diH2O)
• * Can use 0.5X TBE buffer instead to prolong supply of TBE buffer
3. Gel casting trays, combs, and box
4. Loading buffer
5. GelStar Stain
6. Samples to be loaded
7. DNA ladder: 1/20 100bp ladder or 1/20 1000bp ladder (depends on the size of desired product)
• Can add dye directly into the aliquot
8. Graduated cylinder
9. Erlenmeyer flask
10. Parafilm
11. Pipetman of appropriate size

Steps:

1. Pour a 0.8% agarose gel. Weigh out 0.8g of agarose and transfer to a 250mL Erlenmeyer flask.
2. Add 100mLs of 1X or 0.5X TBE buffer and swirl gently to disperse the agarose.
3. Plug flask lightly with a scrunched paper towel, then start by microwaving on high power for 30 seconds. Continue at your own discretion - make sure agarose is completely dissolved.
• *The dissolving step is a fine line between boiling your sample enough to dissolve your material, but not boiling it too much so that liquid starts to evaporate.
4. Allow the solution to cool to 60C by incubating in a 60C waterbath for about 10 minutes.
5. Add GelStar Stain (1/10000, e.g. 5uL/50mL) and swirl to mix.
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• *In this case, add 10uL.
6. Remove casting tray and use the thicker tape to tape the sides of the casting tray.
• Make sure it is sealed tightly so no liquid will escape (especially near the base)
7. Pour into gel casting tray and insert comb.
• *Make sure liquid is not too hot when it is being poured - can melt the tape and leak
• *Gel will take approximately 20 minutes to set.
• *Make sure there are no air bubbles.
8. After gel has solidified, remove combs. Fill box with 1X or 0.5X TBE (if only taking a picture of the gel, TBE can be reused 2 or 3 times, but use new TBE if performing gel extraction).
• *Fill box with 1X if you used 1X to dissolve agarose; fill box with 0.5X if you used 0.5X to dissolve agarose
9. Load samples and ladder into wells.
• *Quick and dirty method of loading samples:
• Pipet 2uL of loading dye onto the parafilm N times with enough spacing inbetween, where N is the number of samples
• Pipet 10uL of sample, touch loading dye on parafilm, pipet up and down without pushing to the second stop on the PipetMan to allow dye to mix, load sample into well.
• Repeat until finished loading samples
10. Run gel. The following is based on the machine in the Lagally Lab.
• Machine conditions: 110V, 45min (can be varied depending on number of samples)
• Remember that DNA is negative, so that bottom electrode should be positive, and top should be negative
• Remember to set the middle button to be constant at Volts and not Amps
• If machine indicates error, there may be too much TBE Buffer in the box. Remove some using a 10mL or 25mL pipet.
• Double check that the machine is running by making sure bubbles are rising (at the top of the box)
11. Remove gel and take to transilluminator/imager on the 3rd floor of MSL, right across from the elevator.
• Wipe down transilluminator surface with water
• Place gel on surface and follow instructions on the door of the transilluminator/imager
• Save file under iGEM in the appropriate folder
• Print picture. *Don’t label the bands directly on the picture
12. Cross-reference sample bands with ladder bands to determine band size.
13. Rinse gel box a few times with water, making sure there are no gel bits. Dry with paper towel and return to original place.

BioBrick Restriction Digests

Supplies Needed:

• Restriction digest supermix (5*n μl of Buffer 2, 0.5*n μl of BSA, 37*n μl of ddH2O where n = # of 50uL reactions) stored at -20°C
• Enzymes
• DNA

Steps:

1. Grab an ice bucket and get some ice.
2. Retreive the appropriate digestion enzyme and digest supermix from -20 fridge and place on ice bucket.
3. Thaw restriction digest supermix (42.5μl aliquots).
4. Add 0.3-0.5μg DNA (1-5uL)
• *Can add up to 10uL of DNA if [DNA] is very low or if stuff doesn’t work
5. Add 1uL of each restriction enzyme.
6. (a). Incubate at 37°C for 2 hours. OR
7. (b). Digest overnight by adding 0.5uL of each restriction enzyme.
8. Return the enzyme and and supermix into the -20 fridge.
9. Discard the ice in the sink.

BioBrick Ligations

Supplies Needed:

• T4 DNA Ligase
• Ligase buffer (1uL aliquots)
• diH2O
• Insert and vector DNA (of approximately known concentration)

Note: Label. Steps:

1. 1. Grab an ice bucket and get some ice.
2. Retrieve ligase and ligation buffer from -20 fridge and place on ice bucket.
3. Calculate insert/vector amounts. Various ratios have been recommended, most commonly 3:1 or 6:1 (molar ratio). Add the calculated insert volume amounts. For simplicity, you can use 1 uL of vector volume as a basis of calculations and then scale up or down.

   Formula: 1 insert mass (ng) = ratio x (insert length/vector length) x vector mass (ng)

   Formula 2: volume of insert (uL)= (total plasmid length/insert length)* insert mass /plasmid concentration

   Example: For a 150bp insert and a 2200bp vector, at a ratio of 6:1: insert mass = 9/22 * vector mass. If you add 1 uL of vector at 30 ng/uL, you would need 9/22*30 of insert, 12.27 ng. If the plasmid DNA concentration were 60 ng/uL and the total plasmid length were 2000, about 3 uL of DNA is needed (2000/150 * 12.27 / 60).

4. Add 1uL ligase buffer (vortex, and make sure it still smells like wet dog). During the process of using the ligase buffer, minimize freeze-thaws.
5. Add sdH2O to 9.5uL and vortex.
6. Add 0.5uL T4 ligase.
7. Incubate at 16°C for at least 1 hour (leave overnight after transforming in case it needs to go for a bit longer).
8. Throw away ice.

Note: Vortexing causes beads of liquid to stick on the walls of the microcentrifuge tube. Spinning in a centrifuge for a few seconds (even less) will cause the liquid to come back to the bottom. A mixed product is the result.

Making Plates

Supplies Needed:

• Petri dishes
• diH2O
• Autoclavable container (500mL glass bottle)
• Antibiotics (Stock solutions: Amp - 100mg/mL in 50% ethanol, Kan - 50mg/mL in H2O, Chl - 35mg/mL in 100%? ethanol, Tet - 15 mg/ml in 50% ethanol - all stored at -20°C)
• Pipet gun + disposable 25mL pipet
• Tinfoil (if using Tet)
• LB Agar Powder
• Colour scheme: Amp - red, Kan - blue, Chl - black, Tet - green

Steps:

1. Calculate amount of media needed (~20mL/plate).
2. Pour 400mL of diH2O into autoclavable container, make sure you remove any autoclave tape on the bottle.
3. Add LB Agar powder (check amount on stock container).
4. Mix as well as you can by swirling and gentle shaking.
5. Autoclave.
• *Mark bottle with autoclave tape, make sure the lid is loose*
• *Follow directions on autoclave*
• *Set to 30 min*
• *Before removing from autoclave make sure the pressure is at 0*
• *Use heat-resistant gloves to remove from Autoclave*
1. Cool until you can hold your wrist against the bottle for 5 seconds without pain.
2. Add antibiotics (final concentrations: A-100-150μg/mL, K-50μg/mL, C-25μg/mL, Tet-10-15μg/mL); if using recommended stock solution concentrations, add 1μL of antibiotic stock per 1mL of media.
3. Swirl gently to mix antibiotics
• *Steps 9-11 are to be done in the Biosafety cabinet in the biohazard room*
• *Ensure the fan and light is on*
• *Ensure no-one else is currently using the Biosafety cabinet*
• *Ethanol everything (unopened plates package, Pipet gun, Pipets, Your arms up to the elbow, bottle of LB Agar) before bringing them inside the Biosafety cabinet*
4. Quickly pipet into plates.
• *Avoid bubbles*
• *Save the plate sleeve, don’t remove from cabinet, must remain sterile*
• Leave to dry 20-30 min before using. Alternatively, leave to solidify 20-30 min before spread plating with appropriate volume of antibiotic.
• For storage, place back in plate sleeve, label according to the colour scheme. If using a light-sensitive antibiotic (Tet), wrap in foil as well.
• *Turn off the light and fan in the biohazard cabinet*
• Keep at 4°C.
• Notes: Ampicillin degrades quickly, so don't make plates more than 2-3 weeks in advance and never leave them at 37°C for more than 16 hours. Kanamycin can last 2-3 months. I don't know about chloramphenicol and tetracycline (not that stable, use like Amp), but they seem to be quite stable as well. Tetracycline is light sensitive.