Team:Manchester/Protocols
PROTOCOLS
1. Inoculate NEB 5α Escherichia coli into 50 ml of LB and leave at 37°C with shaking.
2. Set up TSS buffer (see the table below).
3. Measure the OD600 of the culture until OD600 = 0.4-0.6.
4. Transfer two tubes and centrifuge at 7000 xg at 4°C for 2 minutes.
5. Resuspend the pellet in each tube in 5 ml of TSS and vortex.
6. Chill the mixture for 15 minutes on ice.
7. Aliquot 200 μl samples to microcentrifuge tubes.
8. Use or store at -80°C.
To transform competent E.coli NEB 5-alpha cells we adapted a protocol provided by Promega.
1. Thaw frozen Competent cells on ice.
2. Add 1-50 ng of DNA in 25 μl of Competent Cells and flick the tube few times.
3. Immediately return the tubes on ice for 30 min.
4. Heat shock cells for 20 seconds in 42°C water bath, followed by 2 min incubation on ice.
5. Add 450μl of SOC medium (RT) to the cells and incubate for 60 min at 37°C with shaking.
6. Plate 100 μl of the transformed cells onto agar plates containing antibiotic. Incubate the plates for 12-14 h at 37°C.
DNA purification by centrifugation
Preparation of the Lysate
1. Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.
2. Add an additional 1.5 ml of bacterial culture to the same tube and repeat Step 1.
3. Add 600 µl of water to the cell pellet, and resuspend completely.
4. Add 100 µl of Cell Lysis Buffer (provided in a kit), and mix by inverting the tube 6 times.
5. Add 350 µl of cold (4–8°C) Neutralization Solution (provided in a kit), and mix thoroughly by inverting.
6. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
7. Transfer the supernatant to a PureYield™ Minicolumn without disturbing the cell debris pellet.
8. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed for 15 seconds.
9. Discard the supernatant, and place the minicolumn into the same Collection Tube.
Washing
10. Add 200 µl of Endotoxin Removal Wash (provided in a kit) to the minicolumn and centrifuge at maximum speed for 15 seconds.
11. Add 400 µl of Column Wash Solution (provided in a kit) to the minicolumn and centrifuge at maximum speed for 30 seconds.
Elution
12. Transfer the minicolumn to a 1.5 ml Eppendorf tube.
13. Add 30 µl of Elution buffer (provided in a kit) directly to the minicolumn matrix and let it stand at a room temperature for 1 min.
14. Centrifuge at maximum speed for 15 seconds to elute the plasmid DNA.
15. Store the eluted DNA at -20°C.
1. Pipette 15 µl of ddH2O to separate tubes.
2. Prepare the LB agar plates with antibiotic.
3. Prepare the PCR tubes with a reaction mix (see the table below).
4. Pick up one colony from the plate and inoculate it into the 15 µl ddH2O tube.
5. Pipette 1 µl of the mixture (step 4) into the prepared reaction mix.
6. Plate 9 µl of the mixture (step 4) on a LB agar plate with antibiotic.
7. Leave the plates at 37°C overnight.
8. Run the reaction in the thermocycler with the following conditions (see the table below).
9.Check the product on a gel.
1. Make the reaction mix on ice (see the table below).
2. Incubate the reaction at 37°C for 1h and heat shock at 80°C for 5-10 min.
3. Load the product on a gel to check if the reaction has worked.
DNA purification by centrifugation
Dissolving the gel slice
1. Following electrophoresis, cut the DNA band from gel and place the gel slice in a pre-weighted 1.5 ml microcentrifuge tube.
2. Weight the microcentrifuge tube with a gel slice.
3. Add 10 µl Membrane Binding Solution (provided in a kit) per 10 mg of a gel slice. Vortex and incubate at 55°C water bath until gel slice is completely dissolved (it takes around 15-20 min).
Binding of DNA
4. Insert SV minicolumn into Collection Tube.
5. Transfer dissolved gel mixture into minicolumn assembly. Incubate at room temperature for 1 minute.
6. Centrifuge at 16,000xg for 1 minute.
7. Return the flowthrough to the column and repeat step 6. Repeat this step twice.
8. Then discard the flowthrough and reinsert minicolumn into Collection Tube.
Washing
9. Add 700 µl Membrane Wash Solution (provided in a kit) to the column. Centrifuge at 16,000 xg for 1 minute. Discard the flowthrough and reinsert Minicolumn into Collection Tube.
10. Repeat the step 9 with 500 µl Membrane Wash Solution and centrifuge at 16,000 xg for 5 minutes.
11. Discard the flowthrough and re-centrifuge the column assembly for 1 minute.
Elution
12. Carefully transfer Minicolumn to a clean 1.5 ml Microcentrifuge tube.
13. Add 30 µl of nuclease free water to the minicolumn. Incubate in the water bath (55°C) for 5 minutes and then centrifuge at 16,000 xg for 1 minute.
14. Discard the minicolumn. Store the eluted DNA at -20°C.
The following protocol is adapted from New England BioLabs protocol
1. Mix all the components on ice (see the table below), if you do more than 25 µl of a reaction, add bigger volumes respectively and distribute the mix into separate PCR tubes (up to 25 µl).
2. Gently mix the reaction, spin down to collect all the liquid to the bottom of the tube.
3. Put the samples in already preheated (98°C) thermocycler. Run the PCR with the following conditions (see the table below).
4. Run an agarose gel electrophoresis to check if the PCR has worked.
DNA purification by centrifugation
Processing PCR Amplifications
1. Add an equal volume of Membrane Binding Solution to the PCR amplification.
Binding of DNA
2. Insert SV minicolumn into Collection Tube.
3. Transfer dissolved gel mixture into minicolumn assembly. Incubate at room temperature for 1 minute.
4. Centrifuge at 16,000xg for 1 minute.
5. Return the flowthrough to the column and repeat step 6. Repeat this step twice.
6. Then discard the flowthrough and reinsert minicolumn into Collection Tube.
Washing
7. Add 700 µl Membrane Wash Solution (provided in a kit) to the column. Centrifuge at 16,000 xg for 1 minute. Discard the flowthrough and reinsert Minicolumn into Collection Tube.
8. Repeat the step 9 with 500 µl Membrane Wash Solution and centrifuge at 16,000 xg for 5 minutes.
9. Discard the flowthrough and re-centrifuge the column assembly for 1 minute.
Elution
10. Carefully transfer Minicolumn to a clean 1.5 ml Microcentrifuge tube.
11. Add 30 µl of nuclease free water to the minicolumn. Incubate in the water bath (55°C) for 5 minutes and then centrifuge at 16,000 xg for 1 minute.
12. Discard the minicolumn. Store the eluted DNA at -20°C.
The following protocol is adapted from Promega
1. Calculate molar ratio for vector and insert DNA using NEBioCalculator (ideal ratio for insert:vector is 3:1).
2. Make the ligation mix (see the table below) on ice.
3. Incubate the reaction at:
Room temperature for 3h or
4°C overnight or
15 °C for 4-18 hours.
The following protocol is adapted from Thermo Fisher.
1. Clean up the PCR product with Wizard SV Gel and PCR Clean-Up System.
2. Calculate the amount of the PCR sample needed for a reaction (it is recommended using a 10:1 molar ratio of insert:vector).
3. Make the reaction mix (see the table below).
4. Incubate the ligation reaction at a room temperature for 5 min.
5. Store the product at -20°C.
1. Make the reaction mix (see the table below) on ice.
2. Add 2.5 µl of the Reaction mix to the Gibson master mix. Add another 2.5 µl of the reaction mix to Nuclease-Free Water (as a negative control). 3. Incubate the reaction at 50°C for 1h. 4. Check the product on a gel.
1. Dissolve dried oligo in the TE buffer to make a 100 μM solution and leave for 10 minutes at a room temperature.
2. Prepare the reaction mix (see the table below).
3. Heat the reaction for 5 min at 86°C in a water bath.
4. Transfer some 86°C water together with samples to a large beaker and let it stand at the room temperature to cool down to 30°C.
5. Make a 100-fold dilution with annealed oligos to target 0.1 μM.
1. Autoclave 2 bottles each of 950 ml of M17 broth and M17 agar.
2. Add 50 ml of 10% glucose solution to the 950 ml of M17 broth and M17 agar. M17 now could be labeled as 0.5% GM17.
3. Add Chloramphenicol (to the final concentration of 34 µl/ml) to GM17 broth and GM17 agar.
4. Pipette 20 ml of GM17 in two 50 ml falcon tubes (one for L.lactis and one as a negative control).
1. Weight 0.34 g of Chloramphenicol.
2. Pour 10 ml of 98% ethanol and dissolve the powder completely.
3. Keep the stock at -20°C.
1. Weight 0.5 g of Kanamycin.
2. Pour 10 ml of ddH2O and dissolve completely.
3. Prewet a 0.2 µm syringe filter by drawing through 5-10 ml of sterile H2O and discard water.
4. Sterilize Kanamycin Stock through the prepared 0.2 µm syringe filter.
5. Keep the stock at -20°C.
Day 1
1. Set up the overnight cultures by inoculating the culture into 10 ml of LB with antibiotic.
Day 2
2. Make a 100-fold dilution with each overnight culture in LB+antibiotic (to the final volume of 50 ml) in an Erlenmeyer flask. Incubate the new diluted cultures at 37°C with shaking.
3. Grow the cells in the incubator until they reach Abs600 of 0.4-0.6.
4. Induce the samples with an inducer and grow for 5h at 25°C.
5. Take 1 ml aliquots at different time points (e.g. 0h, 3h, 5h) to monitor the protein expression over time.
6. Make dilutions with aliquots to target OD600 = 1.0 in 1 ml of LB with an antibiotic.
7. Centrifuge the samples for 5‑10 min at 14,000×g and 4°C.
8. Discard the supernatant and resuspend cells in 50 µl of 2x SDS-PAGE dye (0.25 M Tris, 30 % (v/v) glycerol, 10 % (w/v) SDS, 10 mM DTT, 0.05 % (w/v) bromophenol blue).
9. Boil the samples at 95°C for 20 min.
10. Store the product at 4°C.
Day 3
11. Run two same gels; load 7μl of each aliquot and run on 4-20% gradient gels at 200 V for 38 min.
12. Visualise one gel with Quickblue stain.
13. Use the other gel for the Western blot.
1. Run samples on an SDS PAGE of choice (recommend at 200 V for 45 mins).
2. Transfer protein from the gel to the membrane at 30 V for 60-90 mins. Assemble as shown in diagram below:
3. Immerse blotted membrane in 5 mL 1X iBind™/iBind™ FD Solution.
4. Dilute antibodies right after wetting the membrane.
5. Place the iBind™ Card on the stage and pipette 5 mL of 1X iBind™/iBind™ FD Solution evenly across the Flow Region.
6. Add 1 mL of 1X iBind™/iBind™ FD Solution so that it pools at the center of the membrane region on the iBind™ Card.
7. Place the membrane on top of the pooled solution with the protein-side down, and the low molecular weight region closest to the stack.
8. Use the Blotting Roller to remove any air bubbles.
9. Close the lid and add solutions sequentially to the iBind™ Wells starting with Well 1.
10. Place the window cover over the viewing window, and close the well cover. Incubate for 2.5h to overnight.
11. Rinse the membrane in water and proceed to immunodetection protocol.