Team:Bulgaria/PROTOCOLS
Miniprep Plasmid Purification
iGEM Bulgaria used the GeneJET Plasmid Miniprep Kit from Thermo Scientific for all plasmid isolations.
1. Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.
PureLink® Quick Gel Extraction and PCR Purification Combo Kit
iGEM Bulgaria used the PureLink® Quick Gel Extraction and PCR Purification Combo Kit from Thermo Scientifir for all reaction clean-ups, PCR clean-ups and agarose gel extraction procedures.
Purifying PCR fragments or reaction clean-up
1. Combine. Add 4 volumes of Binding Buffer (B2) with isopropanol to 1 volume of a PCR sample (50–100 uL). Mix well.
2. Load. Pipet the sample into a PureLink® Clean-up Spin Column in a Wash Tube. Centrifuge the column at >10,000 × g for 1 minute. Discard the flow-through.
3. Wash. Re-insert the column into the Wash Tube and add 650 uL Wash Buffer (W1) with ethanol. Centrifuge the column at >10,000 × g for 1 minute.
4. Remove ethanol. Discard the flow-through and place the column in the same Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes.
5. Elute. Place the column into a clean 1.5-mL Elution Tube. Add 50 uL Elution Buffer (E1) to the column. Incubate the column at room temperature for 1 minute. Centrifuge the column at maximum speed for 1 minute.
6. Store. The elution tube contains the purified PCR product. Store the purified DNA at 4°C for immediate use or at -20°C for long-term storage.
Purifying DNA from Gels Using a Centrifuge
1. Excise. Use a clean, sharp razor blade to excise a minimal area of gel containing the DNA fragment of interest.
2. Weigh. Using a scale sensitive to 0.001 g, weigh the gel slice containing the DNA fragment.
3. Solubilize. Add Gel Solubilization Buffer (L3) to the excised gel in a tube as indicated in the table. Incubate the tube at 50°C for 10 minutes (or longer for large gel slices and high concentration gels), and invert the tube every 3 minutes. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes. Add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
| Gel | Tube | Buffer L3 Volume: |
|---|---|---|
| ≤ 2% agarose | 1.7-mL polypropylene | 3:1 |
| > 2% agarose | 5-mL polypropylene | 6:1 |
4. Load. Pipet the dissolved gel piece onto a column inside a Wash Tube. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Note: The column reservoir capacity is 850 uL. Use 1 column per 400 mg of agarose gel.
5. Wash. Add 500 uL Wash Buffer (W1) containing ethanol to the column. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes. Discard the flow-through.
6. Elute. Place the column into a Recovery Tube. Add 50 uL Elution Buffer (E1) to the column. Incubate the tube for 1 minute at room temperature. Centrifuge the tube at >12,000 × g for 1 minute.
7. Store. The elution tube contains the purified DNA. Store the purified DNA at 4°C for immediate use or at -20°C for long-term storage.
Oligo annealing and ligation protocol for gRNAs
1. Add 1,5 ul Oligo1 (100 uM stock) and 1,5 ul Oligo2 (100 ul stock) to 500 ul oligo annealing buffer.
2. Incubate for 5' at 95°C.
3. Cool down in 500-800 ml boiling water to RT.
4. Use 4 ul or 8 ul for ligation with 200 ng linearized vector.
5. Store at -20°C.
Ligation
4 ul oligos (16 ng) or 8 ul
200 ng plasmid
2 ul 50% PEG 4000
2 ul 10x T4 ligase buffer
1 ul T4 ligase 5U/ul
Oligo annealing buffer
1 mM Tris pH 7.5
50 mM NaCl
1 mM EDTA
Ligation
1. Add the following reagents to a PCR tube: Reagent Amount 5X Ligase Reaction Buffer Vector DNA 50-100 ng Insert DNA (1:1 or 3:1 ratio) ExpressLink™ T4 DNA Ligase 5 units (in 1 uL) autoclaved distilled water to 20 uL.
2. Mix gently. Centrifuge briefly to bring the contents to the bottom of the tube.
3. Incubate at room temperature for 5 - 15 minutes.
4. Use 2 uL of the ligation reaction to transform 100 uL of electrocompetent cells.
Glycerol Stocks
1. Pick a single colony of the clone off of a plate and grow an overnight in the appropriate selectable liquid medium (e.g., LB amp).
2. Make a label (clone ID # and date) for the construct.
3. Add 0.5ml of the o/n culture to 0.5ml of 80% sterile glycerol in the sterile screw cap microcentrifuge tube.
4. Screw a lid onto the tube and write the clone ID # on the lid of the tube.
5. Vortex.
6. Freeze the glycerol stock at –80°C.
7. To streak out from a glycerol stock:
1. Determine the location (-80°C tower #, box #, row #) of the construct.
2. Take the tube to the place that you intend to streak the clone out.
3. Flame a metal inoculating loop until it is red hot.
4. Scrape off a portion from the top of the frozen glycerol stock and streak it onto your plate.
5. Return the construct to the –80°C.
gBlock Fragment Amplification
1. Phusion® DNA Polymerase amplification reaction
Nuclease-free
H2O Adjust to final 50 uL
5X Phusion HF
10 uL 10 mM dNTPs
1 uL 10 uM Forward Primer
2.5 uL 10 uM Reverse Primer
2.5 uL gBlocks® Gene Fragments
0.1–1.0 ng Phusion® DNA Polymerase 0.5 uL
Total volume 50 uL
2. PCR program:
Initial denaturation: 98°C, 30 seconds
Cycle start:
Denaturation 98°C, 10 seconds
Annealing 60°C, 30 seconds
Extension 72°C, 20 seconds per kb
Cycle end (25-35 cycles)
Final extension 72°C, 5 minutes
Hold 4°C
TSS Competent Cells Preparation and Transformation
Materials:
- dimethyl sulfoxide (DMSO)
- polyethylene glycol 6000 (PEG 6000)1
- 1M MgCl2 stock solution
- 1M MgSO4 stock solution
- LB medium (liquid and 1,5% agar)
- SOC liquid medium2
- antibiotic stocks
Equipment:
- Orbital Shaker at 37°C
- Centrifuge (equipped with rotor for 50 ml Falcon tubes with up to 2700 g support)
- Spectrophotometer
- Thermoblock
Consumables:
- 50 ml Falcon Conical Centrifuge Tubes
- 50 ml sterile Erlenmeyer flask3
- 500 ml sterile Erlenmeyer flask
Protocol
Competent cells preparation
Day 1:
1. Inoculate 5 ml of liquid LB medium with E. coli DH5alfa in a 50 mL Erlenmeyer flask and culture overnight at 37°C with shaking at 250 rpm.
2. Prepare 1x TSS buffer (if you do not have one at -20°C)
To make 20 ml:
• 2 g PEG 60001
• 0.3 ml 1M MgCl2
• 0.3 ml 1M MgSO4
• add LB media to 19 ml and mix until PEG is dissolved
• Filter sterilize (0.22 um filter)
• add 1 ml DMSO (it is sterile by itself)
• Store at at -20°C (should be good for a few months)
Day 2:
1. Prepare 50 ml of liquid LB medium in a sterile 500 ml Erlenmeyer flask.
2. Seed 0.25 ml from the overnight culture into the 500 ml flask and incubate at 37°C with 250 rpm shaking.6
At this point you should take out the 1x TSS buffer, melt it and then store it on ice until it is needed. Additionally, you have to cool down your centrifuge to 2-4°C and put one 50 ml Falcon tube on ice.
3. Cultivate until the optical density of the culture at 600 nm wavelength is 0.3 to 0.4 (0.3 - OD600 - 0.4)7
TSS buffer should be chilled by this point.
4. Once the proper optical density has been achieved, transfer 50 ml of the culture into one 50 ml Falcon Conical Centrifuge Tube and centrifuge it under 2,700xg for 10 min at 2-4°C.
5. Resuspend the cell pellet in 1 ml of pre-chilled 1x TSS buffer with gentle pipetting (use blue tip). Distribute 100 ul of TSS suspended cells to labeled and pre-chilled 1.5 ml Eppendorf tubes while ensuring the cells remain well mixed.
Cells can be used immediately, or stored at -80°C.
Competent cells transformation
1. Thaw TSS cells on ice (if they are not used immediately after preparation) . Thaw some SOC liquid medium.
2. Add DNA, pipette gently to mix (if you are adding small volumes like ~ 1 ul, be careful to mix the culture well). Do not use more than 10 ul sample (1/10 of the cell volume). .
3. Incubate for 30 minutes on ice.
4. Heat shock the cells for 45 seconds at 42°C.
Note: According to the original TSS paper, this step is optional and may actually reduce transformation efficiency. Nevertheless, in our optimization experiments the term shock step increased the observed transformation efficiency for DH5alfa ~ 100 fold (measured when transformed with 300 pg pSB1C3).
5. Incubate cells on ice for 2 min.
6. Add 0.9 ml SOC medium at room temperature.
7. Incubate for 1 hour at 37°C on shaker.
8. Spread 50 ul onto a pre warmed (37°C) LB plate with appropriate antibiotic.
9. Grow overnight at 37 °C.