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Revision as of 13:46, 14 August 2017
Experimental Protocols
Visit Lab Book page for detailed experimental procedures (including trials and errors) undergone by us.
Materials (consumables):
LB broth (Luria Bertrani medium = rich media to grow bacteria)
TSS buffer (to prepare chemically competent cells)
S.O.C. medium (helps obtain the maximal transformation efficiency)
LB agar (gel where bacteria can grow)
Chloramphenicol (CAL) at stock concentration 25mg/ml
Preparation of chemical competent cells:
- Inoculate DH5α cells into 50mL LB and incubate at 37°C
- Monitor growth every 30 min by measuring optical density at 600nm (OD600) until it reaches OD600 = 0.4-0.6
- Harvest cells and prepare using 'TSS competent E.coli protocol' shown below:
LB Agar plates preparation:
- Prepare LB containing chloramphenicol (CAL) (at 25μg/ml)
- Melt LB in microwave (defrost setting for 15 min)
- Cool LB by running cold water over
- Stock of 25mg/ml CAL → so add 400μl CAL to 400ml LB
- Pour plates (in fume hood) and allow to solidify
Chemical Transformation:
- Add 1μl of DNA to 50μl of competent cells, mix well and place on ice for at least 30 min
- Heat shock cells at 42°C for 30 seconds, followed by 2 min incubation on ice
- Add 450μl of SOC medium to the cells and incubate for 45 min at 37°C (to allow protein expression (particularly the antibiotic resistance gene)
- Plate and spread (glass spreader sterilised over a flame and in ethanol) 50, 100, and 200μl of the cells into the agar plates made previously
- Incubate at 37°C for 2 hours
Materials (consumables):
Dimethyl sulfoxide (DMSO)
Polyethylene glycol (PEG)
MgCl2 stock solution
LB or SOC liquid medium
Ice
Methods:
- Inoculate DH5α E. coli cells into 50ml LB broth and incubate at 37°C
- Prepare the TSS buffer while waiting for the culture to grow and place it on ice (see: Table below)
- Monitor growth of the culture every 30 min by measuring the optical density at 600nm wavelength (OD600) until it reaches OD600 = 0.4-0.6 (takes approximately 2-3 hours)
- Once the proper optical density has been achieved, take 100ml of culture and centrifuge under 2.700xg for 10 min at 4°C
- Resuspend each tube in 5ml of pre-chilled TSS buffer with gentle vortexing
- Chill TSS suspended cells on ice for 15 min
- Aliquot 200μL of TSS suspended cells while ensuring the cells remain well mixed
- Cells can be used immediately or stored at -80°C
TSS Buffer Composition
| Component | Stock (M) | Amount |
|---|---|---|
| MgCl2 | 2 | 0.300ml |
| DMSO | - | 1ml |
| PEG (3350 or 8000) | - | 2g |
| LB Medium | to final volume of 20ml | to final volume of 20ml |
Materials (consumables):
2x Q5 Master Mix
10 µM forward primer
10 µM reverse primer
DNA template
Nuclease-free water
Methods:
- Gently mix the reaction from the components listed in the table below and place on ice
Note: addition of reagents are done in the following order to prevent degradation of primers: nuclease-free water, both primers, DNA, and then Q5 mix - When necessary, collect all liquid to the bottom of the PCR tube by spinning for a short time
- Transfer the PCR tube from ice to a PCR machine and begin thermocycling
PCR Reaction Components
| Component | 25µl Reaction | 50µl Reaction | Final concentration |
|---|---|---|---|
| Q5 High-Fidelity 2X Master Mix | 12.5µl | 25µl | 1X |
| 10µM Forward Primer | 1.25µl | 2.5µl | 0.5µM |
| 10µM Reverse Primer | 1.25µl | 2.5µl | 0.5µM |
| Template DNA | variable | variable | < 1,000ng |
| Nuclease-Free Water | to 25µl | to 50µl |
Thermocycling
The PCR machine should be set to run the following steps:
| Step | Temperature (°C) | Time |
|---|---|---|
| Initial denaturation | 98 | 30 seconds |
| 30 cycles | 98 (denaturation) 63 (annealing) see Note 1 72 (extension) |
5 seconds 30 seconds 27 seconds per kb |
| Final extension | 72 | 2 minutes |
| Hold | 10 | - |
Note 1: The NEB Tm calculator should be used to determine the annealing temperature when using the Q5 Master Mix: http://tmcalculator.neb.com/#!/
Materials (consumables):
1% TAE Buffer
Agarose powder
SYBR Safe
Loading dye
DNA ladder
Make 1% agarose gel:
- Prepare 1% TAE agarose gel: dissolve 1g of agarose into 100ml of TAE buffer in a conical flask
- Warm in microwave for 1 min at max power
- Remove flask from microwave with care, swirl gently and cool under running tap
- Add 5µl of SYBR Safe
- Prepare a casting tray with suitable comb
- Pour to cool mixture into the casting tray and wait 15 min until it solidifies
Run gel:
- Add 5µl of PCR solution and 1µl 10x loading dye
- Load 6µl of DNA ladder alongside and all samples (Do not forget to add dye to ladder too) - NEB 1kb ladder used
- Run gel at 100V for 45 min
- Visualise gel on a transilluminator (SYBR Safe binds DNA and fluoresces under UV light)
Materials (consumables):
Restriction Enzyme: NEB enzyme finder to determine the restriction enzymes
10X Buffer: NEB double digest finder to determine the buffers that are required
Plasmid DNA
Nuclease-free water
Methods:
- Incubate the digestion reaction (see components below) for 1 hour at 37°C, then at 65°C for 20 min (to heat-inactivate the enzymes)
Restriction Digestion Mix
| Component | 50µl Reaction | Final concentration |
|---|---|---|
| 10X Buffer | 50µl | 1X |
| DNA | will vary | 250ng |
| Restriction Enzymes | 1µl (of each enzyme) | 10U |
| Nuclease-free water | to 50µl |
Materials (consumables):
T4 Ligase Buffer
T4 Ligase
Vector DNA
Insert DNA
Nuclease-free water
Ice
Methods:
- Allow the buffer to defrost on ice
- Calculate molar ratio for vector and insert DNA using NEBioCalculator (NEBioCalculator) (ideal ratio for insert:vector is 3:1)
- Make up ligation reaction as below
- If using T4 ligase: incubate at room temp for 1 hour
- NB: different ligases (eg. Quick Ligase) will require different incubation times
Ligation Reaction Composition
| Component | 10µl Reaction | Final concentration |
|---|---|---|
| 10X T4 Ligase Buffer | 1µl | 1X |
| Vecor DNA | vary | |
| Insert DNA | vary | |
| Nuclease-free water | to 10µl |
Materials (consumables):
Ethanol (96-100%)
Buffer PE
Buffer PB
Buffer EB
pH indicator I
Sodium acetate
QIAquick column
Loading dye
Notes before starting:
- Add ethanol (96-100%) to buffer PE before use (see bottle label for volume)
- All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature
- Add 1:250 volume pH indicator I to Buffer PB. The yellow color of Buffer PB with pH indicator I indicates a pH of ≤7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I. Do not add pH indicator I to buffer aliquots
Methods:
- Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10μl 3M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
- Place a QIAquick column in a provided 2 ml collection tube
- To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 seconds until all the samples have passed through the column. Discard flow-through and place the QIAquick column back in the same tube
- To wash, add 0.75ml Buffer PE to the QIAquick column and centrifuge for 30–60 seconds. Discard flow-through and place the QIAquick column back in the same tube
- Centrifuge the QIAquick column once more in the provided 2ml collection tube for 1 min to remove residual wash buffer
- Place each QIAquick column in a clean 1.5ml microcentrifuge tube
- To elute DNA, add 50μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge
- If the purified DNA is to be analyzed on a gel, add 1 volume of Loading dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel
Materials (consumables):
Overnight culture
Buffer P1
Buffer P2
Buffer N3
Buffer PE
Sterile dH2O (MiliQ) water
Methods:
- Spin down the overnight cultures at 10000rpm for 10 min. Discard supernatant into virkon
- Resuspend the pelleted bacteria with 250μl of Buffer P1 (stored in fridge), and transfer the resuspended bacteria into a fresh 2ml eppendorf
- Add 250μl of Buffer P2 to the 2mL Eppendorf with bacteria and mix gently. Sample should turn blue (indicates cells have lysed)
- Incubate for 5 min at room temperature (do not exceed 5 min or plasmid will begin to degrade)
- Add 350μl of Buffer N3 and mix gently. Sample should be colourless and contain a white precipitant
- Centrifuge samples at 14,000rpm for 10 min using a table top centrifuge
- Transfer 750μl of the supernatant to a column placed on a 1.5ml Eppendorf tube (discard white precipitate). Centrifuge at 11,000rpm for 1 min using a table top centrifuge
- Discard the flow-through. Place column onto new eppendorf tube and add 750μl PE buffer (with added ethanol to the stock buffer if not already done so). Incubate at room temperature for 5 min. Centrifuge at 13,000rpm for 30 seconds
- Transfer column to fresh eppendorf. Centrifuge at 13,000rpm for 2 min (dry out)
- Transfer column into fresh eppendorf. Add 30μl of sterile dH20 (MiliQ) (add directly onto column to ensure water pushes DNA through) and incubate for 5 min at room temperature
- Centrifuge at 11,000rpm for 1 min. Do not discard this flow-through. This contains the extracted plasmid
Materials (consumables):
Buffer QG
Buffer PE
Buffer PB
3M Sodium Acetate
Sterile dH2O (MiliQ) water
Isopropanol
Methods:
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
- Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100mg gel ~ 100μl). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG
- Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow
- Add 1 gel volume isopropanol to the sample and mix
- Transfer 750µl of supernatant to a column placed on a 1.5ml eppendorf. Centrifuge at 11,000 rpm for 1 min
- Discard flow through. Place column in a new Eppendorf tube. Add 500µl of PB buffer and centrifuge column at 13,000 rpm for 30 seconds
- Discard flow through. Place column in a new eppendorf. Add 750µl of PE buffer (check ethanol has been added, see: PCR Purification). Incubate at room temp for 5 mins, then centrifuge at 13,000 rpm for 30 seconds
- Transfer column into fresh Eppendorf. Centrifuge at 13,000 rpm for 2 mins
- Transfer column to new eppendorf. Add 30µl of MiliQ water and incubate for 5 mins at room temperature
- Final centrifuge at 11,000 rpm for 1 minute
- Test sample using Nanodrop (see below)
Nanodrop: calculate DNA concentration in sample
- Load 1μL of MiliQ water to the Nanodrop and blank. Clean and load another 1μl of MiliQ water, measure and proceed only if clean (absorbance is zero at all measured wavelengths)
- Load 1μl of sample and measure DNA concentration
- Check A260/280 ~ 1.8 and A260/230 ~2.0 (pure DNA sample should have values close to these numbers)
Materials (consumables):
coming soon...
Method:
- coming soon...
Materials (consumables):
coming soon...
Method:
- coming soon...
Materials (consumables):
coming soon...
Making slides:
- coming soon...
Fluorescence Microscopy:
- coming soon...
Software settings:
coming soon...
Method:
- coming soon...
