Team:Hong Kong HKU/Experiments
|
Experiments & Protocols
Preparation of competent cells
(i) Streaking MG1655 plate
- a MG1655 plate with no antibiotics provided by Professor Jin
- a LB agar plate without antibiotics
- LB broth
1. Add 50 uL of LB broth to the new LB plate without antibiotics.
2. Pick 1 colony from the MG1655 plate and transfer it to the LB plate.
3. Streak the LB plate with the colony.
4. Incubate the plate overnight at 37 degree Celsius.
(ii) Preparation of 200-mL of LB
- 1-L conical flask
- LB powder (20gm/L)
- ddH2O
1. Rinse the conical flask with ddH2O 3 times.
2. Dissolve 4 g of LB powder with 200 mL of ddH2O in the flask.
3. Autoclave the solution for 3 hours.
(iii) Preparation of 100 mL of 0.1M CaCl2
- CaCl2-2H2O
- ddH2O
- reagent bottle
1. Weight 1.47 g of CaCl2-2H2O (MW: 147.02 g/mol).
2. Dissolve CaCl2-H2O into 100 mL of ddH2O in the reagent bottle.
3. Autoclave the solution for 3 hours.
(iv) Preparation of 0.1M CaCl2/15% glycerol
- CaCl2-2H2O
- ddH2O
- 100% glycerol
- reagent bottle
1. Weight 1.47 g of CaCl2-2H2O (MW: 147.02 g/mol) and add it to the reagent bottle.
2. Add 25 mL of ddH2O to dissolve the solid.
3. Add 15 mL of 100% glycerol to the solution and add 25 mL of ddH2O to dissolve glycerol.
4. Add ddH2O again until the total volume reaches 100 mL.
5. Autoclave the solution for 3 hours.
(v) Picking colony
- LB broth
- a 25-mL centrifuge tube
- the MG1655 plate streaked previously
1. Add 5 mL of LB broth to the centrifuge tube labelled.
2. Pick a colony from a plate and transfer it to the tube.
3. Incubate the tube of cells overnight at 37 degree Celsius.
(vi) Making competent cells
- the MG1655 overnight culture prepared previously
- the 1-L conical flask containing 200-mL LB prepared previosly
- the reagent bottle containing 100 mL of 0.1M CaCl2 prepared previously
- the reagent bottle containing 100 mL of 0.1M CaCl2/15% glycerol prepared previously
1. Add 2 mL of MG1655 overnight culture to 200 mL of LB.
2. Place the flask containing MG1655/LB to the shaker and shake it at 220 rpm for 1.5 hours at 37 degree Celsius.
3. During shaking, measure the OD600 of the overnight culture by diluting 10X with LB.
4. After 1.5-hour incubation, measure the OD600 of MG1655 in LB by transferring 1 mL of cell culture to the cuvette.
5. When the OD600 reaches 0.5-0.6, put the flask on ice and cool it to around 4 degree Celsius for 15 minutes.
6. Transfer the culture to 2 250-mL centrifuge bottles on a balance to ensure equal division to the 2 bottles.
7. Centrifuge the 2 bottles at 3000 rpm at 4 degree Celsius for 10 minutes.
8. Remove the supernatant and resuspend the pellet with 20 mL of 0.1M CaCl2 in each bottle by pipetting up and down, or shaking the bottle gently.
9. Incubate the 2 bottles on ice for 30 minutes.
10. Transfer the culture from the centrifuge bottles to 2 50-mL centrifuge tubes.
11. Centrifuge the 2 tubes at 3000 rpm at 3 degree Celsius for 10 minutes.
12. Remove the supernatant and resuspend the pellet with 4 mL of 0.1M CaCl2/15% glycerol.
13. Divide the culture into aliquots of 100 uL each in Eppendorf tubes. Place the tubes in liquid nitrogen for >5 minutes.
14. Collect and store the tubes of cells in the freezer at -80 degree Celsius.
Test for cell competency / Transformation
- the Competent Cell Test Kit (purified DNA from BBa_J04450 in pSB1C3)
→ 50 pg/uL
→ 20 pg/uL
→ 10 pg/uL
→ 5 pg/uL
→ 0.5 pg/uL
- ddH2O as negative control
- 6 MG1655 competent cells aliquots (100 uL each) in Eppendorf tubes
- 6 LB agar plates with CM30
- LB broth
1. Spin down the DNA tubes from the Competent Cell Test Kit to collect all of the DNA into the bottom of each tube prior to use.
2. The competent cells in Eppendorf tubes were thawed on ice.
3. Pipet 1 uL of DNA into each labelled Eppendorf tube. 6 Eppendorf tubes for 6 different concentrations of DNA.
4. Flick the tubes gently to mix and incubate them on ice for 30 minutes. At the same time, preheat the waterbath to 42 degree Celsius.
5. Heat-shock the cells by placing the Eppendorf tubes into waterbath for 1.5 minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
6. Immediately transfer the tubes back to ice to let the cells recover by incubating them on ice for 3 minutes.
7. Add 1mL of LB broth into each tube and incubate the tubes at 37 degree Celsius for 1 hour. At the same time, label the CM30 plates properly.
8. Pipet 100 uL from each Eppendorf tube onto the appropriate plate, and spread the mixture evenly across the plate.
9. Incubate the plates at 37 degree Celsius overnight or approximately 16 hours. Position the plates so the agar is facing up, and the lid is facing down.
Miniprep
- QIAprep® Spin Miniprep Kit
- 5 mL of bacterial overnight culture
1. Pellet the bacterial overnight culture by centrifugation at 4000 rpm for 3 minutes at room temperature.
2. Mix the provided RNase A solution with Buffer P1 (100-fold).
3. Resuspend the pelleted bacterial cells in 250 uL P1/RNase A and transfer to an Eppendorf tube.
4. Add 250 uL Buffer P2 and mix thoroughly by inverting the tube 10 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
5. Add 350 uL Buffer N3 and mix immediately and thoroughly by inverting the tube 10 times.
6. Centrifuge the tube for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
7. Apply the supernatant from step 6 to the QIAprep spin column by pipetting. Centrifuge for 1 minute and discard the flow-through.
8. Wash the QIAprep spin column by adding 0.5 mL Buffer PB. Centrifuge for 1 minute and discard the flow-through.
9. Add ethanol (96-100%) to Buffer PE so that ethanol makes up with 80% of the solution.
10. Wash the QIAprep spin column by adding 0.75 mL Buffer PE. Centrifuge for 1 minute and discard the flow-through. Transfer the QIAprep spin column to the Eppendorf tube.
11. Centrifuge for 1 minute to remove residual wash buffer.
12. Add 50 uL PCR water to the center of the QIAprep spin column to elute DNA, let stand for 1 minute, and centrifuge for 1 minute.
PCR using Q5 High-Fidelity DNA Polymerase
1. Prepare the gBlock DNA template for a 50 uL reaction. Add 1uL of 10 ng/uL gBlock DNA to 9 uL of PCR water to make up the concentration as 1 ng/uL. 1 ng of DNA template is enough.
2. Add (n mole/10) mL of TE buffer to each oligonucleotide primer to make up the concentration as 10 uM. A final concentration of 0.5 uM in the reaction would show the best result.
3. Assemble all reaction components on ice. Gently mix all the components before use. Collect all liquid to the bottom of the tube by a quick spin if necessary.
| COMPONENT | 50 uL REACTION |
|---|---|
| Q5 High-Fidelity 2X Master Mix | 25 uL |
| 10 uM Forward Primer | 2.5 uL |
| 10 uM Reverse Primer | 2.5 uL |
| Template gBlock DNA | 1 uL |
| DMSO | 1 uL |
| PCR water | 18 uL |
4. If Taq polymerase is used instead of Q5, assemble all reaction components as followed on ice. Gently mix all the components before use. Collect all liquid to the bottom of the tube by a quick spin if necessary.
| COMPONENT | 50 uL REACTION |
|---|---|
| Taq buffer | 5 uL |
| dNTPs | 1 uL |
| 10 uM Forward Primer | 1 uL |
| 10 uM Reverse Primer | 1 uL |
| Taq polymerase | 0.5 uL |
| Template gBlock DNA | 2 uL |
| PCR water | 39.5 uL |
5. Transfer PCR tubes to a PCR machine and begin thermocycling.
| STEP | TEMPERATURE | TIME |
|---|---|---|
| Initial Denaturation | 98 degree Celsius | 30 second |
| 30 cycles | 98 degree Celsius | 10 seconds |
| 50-72 degree Celsius | 30 seconds | |
| 72 degree Celsius | 1.5 minutes | |
| Final Extension | 72 degree Celsius | 2.5 minutes |
| Hold | 4 degree Celsius |
Gel Extraction
- QIAquick Gel Extraction Kit
1. Cut the specific DNA fragments from the agarose gel with a scalpel.
2. Weigh the gel slice in an Eppendorf tube. Add 3 volumes buffer QG to 1 volume gel (100 mg gel to 100 uL).
3. Incubate the tube at 50 degree Celsius for 10 minutes. Vortex the tube every 2-3 minutes to help dissolve gel. The colour of the mixture is yellow.
4. Add 1 gel volume isopropanol to the sample and mix.
5. Place a QIAquick column in s provided 2-mL collection tube. Apply the sample to the QIAquick column and centrifuge for 1 minute. Discard the flow-through and place back the column back into the same tube (binding DNA).
6. Add 750 uL of buffer PE/EtOH to QIAquick column. Let the column stand for 4 minutes and centrifuge for 1 minute. Discard the flow-through and place back the QIAquick column back into the same tube (washing). Centrifuge the QIAquick column in the provided 2-mL collection tube for 1 minute to remove residual wash buffer.
7. Place the QIAquick column into a clean Eppendorf tube.
8. Add 30 uL of PCR water to the centre of the QIAquick membrane to elute DNA. Let the column stand for 1 minute and centrifuge for 1 minute.
HiFi DNA Assembly and Transformation
- NEBuilder HiFi DNA Assembly Master Mix
- Positive control provided: 2 overlapping dsDNA fragments for control assembly
| length (bp) | Conc. (ng/uL) | pmol needed | Mass (ng) | Vol. (uL) | |
| gBlock 1 | 1160 | 19.5 | 0.05 | 35.84 | 1.84 |
| gBlock 2 | 1889 | 10 [stock] | 0.05 | 58.37 | 5.84 |
| gBlock 3 | 1540 | 23.8 | 0.05 | 47.58 | 2.00 |
| gBlock 4 | 2040 | 8.7 | 0.05 | 63.03 | 7.24 |
| gBlock 5 | 723 | 36.5 | 0.05 | 22.34 | 0.61 |
| pSB1C3 | 2070 | 25 [stock] | 0.05 | 63.96 | 2.56 |
1. Set up the following reaction on ice:
| 4-6 Fragment Assembly | Positive Control | |
|---|---|---|
| Recommended DNA Ratio | vector:insert = 1:1 | |
| Total Amount of Fragments | 0.2-0.5 pmols X uL
|
10 uL |
| NEBuilder HiFi DNA Assembly Master Mix | 20 uL | 10 uL |
| Total Volume | 40.09 uL | 20 uL |