Bacteria Culture
1. Prepare LB Media.
To make 400 mL of LB, weigh out the following into a 500 mL glass bottle:
| LB Broth |
LB Agar |
| 4 g NaCl |
4 g NaCl |
| 4 g Tryptone |
4 g Tryptone |
| 2 g Yeast Extract |
2 g Yeast Extract |
|
6 g Agar |
| Add ddH2O to 400 mL |
Add ddH2O to 400 mL |
2. Autoclave
Loosely close the cap on the bottle (Do NOT close all the way or the bottle may explode!) and then loosely cover the entire top of the bottle with aluminum foil. Autoclave and allow to cool to room temperature. Now screw on the top of the bottle and store the LB at room temperature.
3. Antibiotics
When ready to grow your culture, add liquid LB to a tube or flask and add the appropriate antibiotic to the correct concentration.
| Antibiotics Concentration |
| Ampicillin |
150 µg/mL |
| Kanamycin |
20 µg/mL |
4. Inoculation
Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
Incubate bacterial culture at 37°C for 12-18 hr in a shaking incubator.
Mini Prep. (FAVORGEN)
1. Transfer 1~ 3 ml of well-grown bacterial culture to a centrifuge tube
2. Centrifuge the tube at 11,000 x g for 1 minute to pellet the cells and discard the supernatant completely.
3. Add 200 µl of FAPD1 Buffer (RNase A added) to the cell pellet and resuspend the cells completely by pipetting.
• Make sure that RNase A has been added into FAPD1 Buffer when first use.
• No cell pellet should be visible after resuspension of the cells.
4. Add 200 µl of FAPD2 Buffer and gently invert the tube 5 ~ 10 times. Incubate the sample mixture at room temperature for 2 ~ 5 minutes to lyse the cells.
• Do not vortex, vortex may shear genomic DNA. If necessary, continue inverting the tube until the lysate become clear.
• Do not proceed the incubation over 5 minutes.
5. Add 300 µl of FAPD3 Buffer and invert the tube 5 ~ 10 times immediately to neutralize the lysate.
• Invert immediately after adding FAPD3 Buffer will avoid asymmetric precipitation.
6. Centrifuge at full speed (~18,000 x g) for 5 min to clarify the lysate. During centrifugation, place a FAPD Column in a Collection Tube.
7. Transfer the suspernatant carefully to the FAPD Column and centrifuge at 11,000 x g for 30 seconds. Discard the flow-through and place the column back to the Collection Tube.
• Do not transfer any white pellet into the column.
8. Add 400 µl of W1 Buffer to the FAPD Column and centrifuge at 11,000 x g for 30 seconds. Discard the flow-through and place the column back to the Collection Tube.
Restriction enzyme double digestion
Reagent Quantity Final concentration
| Content |
Amount |
| DNA |
1 µg |
| 10X NEBuffer (1X) |
5 µl |
| Restriction Enzyme #1 |
1 µl (10 units) |
| Restriction Enzyme #2 |
1 µl (10 units) |
| ddH2O |
to 50 µl |
1. Enzyme digestion was carried out 1 hour at 37 °C in incubator. (This time could be extended if it does not work well)
Gel electrophoresis
1. Prepare the gel solution by adding 1 g agarose in to100 ml TAE 1X buffer.
2. Heat until all agarose has dissolved completely.
3. Add 10µl EcoDye (BIOFACT) into the cooled solution (at around 60°C), mix the solution evenly through shaking.
4. Pour the solution into a cast and place the corresponding comb on top of the gel cast.
5. Wait for the gel to solidify (about 30 minutes).
6. Transfer the solid gel into the electrophoresis apparatus with TAE 1X buffer submerged.
7. Add 10 µl DNA samples and 5 µl DNA Ladder into cells of the gel.
8. Run the electrophoresis at 110 V for 20 minutes.
9. Visualize the gel under UV light.
Gel Extraction (QIAquick Gel Extraction Kit Protocol)
1. Excise DNA fragment from agarose gel with a clean, sharp scalpel. A 1% regular agarose in 1X TBE is fine.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QX1 to 1 volume gel (100 mg ~ 100 μl). The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg, use more than one column.
3. Incubate at 50oC for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2-3 min during the incubation.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow.
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2-ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
8. Discard flow-through and place the QIAquick column back in the same collection tube.
9. (Optional): Add 0.5 ml of Buffer QX1 to QIAquick column and centrifuge for 1 min.
10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
11. Discard flow-through and centrifuge the QIAquick column for an additional 1 min at ~13,000 rpm.
12. Place QIAquick column into a clean 1.5-ml microfuge tube.
13. To elute DNA, add 50 μl of 10 mM Tris-HCl, pH 8.5 of H2O to the center of the QIAquick column and centrifuge for 1 min at maximum speed. Alternatively, for
increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick column, let stand for 1 min, and then centrifuge for 1 min.
Ligation
| Content |
Amount |
| Vector DNA |
10 ng |
| Insert DNA |
Molar ratio 3:1 |
| 10X T4 Ligase buffer |
2 µl |
| T4 DNA Ligase (added last) |
1 µl |
| ddH2O |
to 20 µl |
1. Gently mix by up and down pipetting
2. Incubate at room temperature for 2 h or at 16 C overnight.
3. Proceed with transformation.
Note: that the ratio of insert to backbone is approximately 3:1. (Total DNA volume should ≦10µl)
All the reactions above require enzyme to be added in the last step.
Transformation
1. Take competent cell from -80°C and place it on ice. DO NOT interrupt.
2. Use separate EP tube for experiment group, negative control and positive control. Operate on ice.
| Group |
Content |
| Experiment |
Competent cell + 10 µl ligation product |
| Positive control |
25 µl competent cell+ 1 µl vector |
| Negative control |
25 µl competent cell+1µl dd H2O |
3. Waite 20 min (still on ice). Preheat the heat bath to 42°C
4. Heat Shock: Heat bath 42°C for 90s.
5. Replace immediately on ice for 2 min.
6. For Recovery, add 500µl antibiotic-free LB. This should be done in the clean Bench.
7. Incubation: under 37 °C, 200~300 rpm for 20 min.
8. Spread the product (competent cell) on the corresponding agar plates. (the petri dishes should be marked).
SDS-PAGE (Bio-Rad)
Casting the Gel
0. Assemble glass plates and spacers in gel casting apparatus
1. Mix the components for the resolving gel as described in the Mini-Protean II protocol.
2. Pour the resolving gel mixture into the gel plates to a level 2 cm below the top of the shorter plate.
3. Pace a layer of DDI H2O over the top of the resolving gel to prevent meniscus formation in the resolving gel.
4. Allow resolving gel to stand 30 min at room temperature.
5. Drain the DDI H2O from top of the resolving gel. Rinse with DDI H2O, drain, and wick any remaining DDI H2O away with a Kimwipe.
6. Mix components for stacking gel.
7. Pour stacking gel solution into gel plates (on top of running gel), so that gel plates are filled. Insert comb to the top of the spacers.
8. Allow gel to stand for at least 1 hr at room temperature, or overnight at 4°C (wrapped in saran wrap).
Preparing Samples
Note: 10 well combs will hold up to 30 µl of prepared sample. 15 well combs will hold up to 20 µl of prepared sample.
Cell Samples
0. Harvest 100 µl of cells at O.D. > 0.6. Decant the supernatant media.
1. Resuspend cells in 20 µl of 2x sample buffer.
2. Incubate tubes in boiling water for 5 min.
3. Centrifuge at 12,000 x g for 30 s.
Solution Samples
0. Place a volume of protein solution (or 1 µl of standard) into a µfuge tube, such hat there is 5-10 µg of protein in the solution.
1. And an equal volume of 2x sample buffer (or 10 µl for standards).
2. Incubate tubes in boiling water for 5 min.
3. Centrifuge at 12,000 x g for 30 s.
Running the Gel
0. Remove comb and assemble cast gel into Mini-Protean II apparatus.
1. Add freshly prepared 1x running buffer (300 ml) to both chambers of the apparatus.
2. Load the prepared samples into the wells of the gel.
3. Run the gel at 100 V until the dye front migrates into the running gel (~15 min), and increase to 200 V until the dye front reaches the bottom of the gel (~45 min).
Staining & Destaining the Gel
0. Remove the run gel from the aparatus and remove the spacers and glass plates. Place the gel into a small tray.
Note: Never us a metal spatula to separate the glass plates.
1. Add ~20 ml staining solution and stain for > 30 min with gentle shaking.
2. Pour off and save the stain.
3. Add ~5 ml destain solution and destain for ~1 min with gentle shaking.
4. Pour off and discard the destain solution. Add ~ 30 ml of destain solution.
5. Destain with gentle shaking until the gel is visibly destained (> 2 hr).
6. Pour off and discard the destain solution.
7. Rinse with DDI H2O. Add ~30 ml DDI H2O and rinse for 5 min with gentle shaking.
8. Dry the gel on the gel dryer at 60°C for 1 hr with a sheet of Whatman filter paper below the gel and a piece of Seran wrap over the gel.
Western Blot (Bio-Rad)
Sample Prep
1. Place the cell culture dish in ice and wash the cells with ice-cold Tris-buffered saline (TBS)
2. Aspirate the TBS, then add ice-cold RIPA buffer (1ml per 100mm dish)
3. Scrape adherent cells off the dish using a cold plastic cell scraper and gently transfer the cell suspension into a precooled microcentrifuge tube
4. Maintain constant agitation for 30 min at 4℃
5. If necessary, sonicate 3 times for 10-15 sec to complete cell lysis and shear DNA to reduce sample viscosity
6. Spin at 16,000×g for 20 min in 4℃ precooled centrifuge
7. Gently remove the centrifuge tube and place it on ice. Transfer the supematant to a fresh tube, also kept on ice, and discard the pellet
8. Remove a small volume (10-20μl) of lysate to perform a protein assay. Determine the protein concentration for each cell lysate.
9. If necessary, aliquot the protein samples for long-term storage at –20℃. Repeated freeze and thaw cycles cause protein degradation and should be avoided.
10. Take 20μg of each sample and add an equal volume of 2X Laemmli sample buffer
11. Boil each cell lysate in sample buffer at 95℃ for 5min
12. Centrifuge at 16,000×g in a microcentrifuge for 1 min\
Protein separation by gel electrophoresis
1. Load equal amounts of protein (20μg) into the wells of a mini (8.6×6.7cm) or midi (13.3×8.7cm) format SDS-PAGE gel, along with molecular weight markers.
2. Run the gel for 5 min at 50V
3. Increase the voltage to 100-150 V to finish the run in about 1 hr.
Transferring the protein from the gel to the membrane
1. Place the gel in 1× transfer buffer for 10-15 min
2. Assemble the transfer sandwich and make sure no air bubbles are trapped in the sandwich. The blot should be on the cathode and the gel on the anode.
3. Place the cassette in the transfer tank and place an ice block in the tank.
4. Transfer overnight in a coldroom at a constant current of 10mA. Transfer can also be done at 100V for 30min-2hr.
Antibody Incubation
1. Briefly rinse the blot in water and stain it with Poceau S solution to check the transfer quality
2. Rinse off the Ponceau S stain with three washes with TBST
3. Block in 3% BSA in TBST at room temperature for 1 hr
4. Incubate overnight in the primary antibody solution against the target protein at 4℃
5. Rinse the blot 3-5 times for 5 min with TBST
6. Incubate in the HRP-conjugated secondary antibody solution for 1 hr at room temperature. The antibody can be diluted using 5% skim milk in TBST
7. Rinse the blot 3-5 times for 5 min with TBST
Imaging and data analysis
1. Apply the chemiluminescent substrate to the blot
2. Capture the chemiluminescent signal using a CCD camara-based imager
3. Use image analysis software to read the band intensity of the target proteins
Stripping and reprobing
1. Warm the buffer to 50℃
2. Add the buffer to the membrane in a container designated for stripping. Incubate at 50℃ for up to 45 min with some agitation
3. Rinse the blot under running water for 1 hr
4. Transfer the membrane to a clean containerm, wash 5 times for 5 min with TBST
5. Block in 3% BSA in TBST at room temperature for 1 hr
6. Incubate overnight in the primary antibody solution (against the loading control protein) at 4℃
7. Rinse the blot 3-5 times for 5 min with TBST
8. Incubate in the HRP-conjugated secondary antibody solution for 1 hr at room temperature
9. Rinse the blot 3-5 times for 5 min with TBST
Imaging and data analysis
1. Apply the chemiluminescent substrate to the blot
2. Capture the chemiluminescent signal using a CCD camara-based imager
3. Use image analysis software to read the band intensity of the target proteins
4. Use the loading control protein levels to normalize the target protein levels.
Reference
Saeidi, N., Wong, C. K., Lo, T., Nguyen, H. X., Ling, H., Leong, S. S., . . . Chang, M. W. (2014). Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Molecular Systems Biology, 7(1), 521-521. doi:10.1038/msb.2011.55
