Team:NCKU Tainan/Experiments
This page is about our protocols, you can find all the experimental process you need.
Enjoy!
Congo red Assay
Materials:
Equipment:
1. Pipette and pipette tips
2. Centrifuge
3. ELISA reader
4. Incubator
5. 96 well plate
6. Centrifuge tube
Consumables:
1. Congo red
2. IPTG
3. LB
4. ddH2O
Protocol:
1. Culture bacteria NOS-DA-BL21, BW25113 csgD, BW25113 control for 2hr.
2. Add IPTG for induce for 12hr.
3. Adjust OD to 1.
4. Add congo red 3μl to 1ml bacteria.
5. Wait 2hr.
6. Centrifuge full speed 2min.
7. Remove supernatant.
8. Add 1 ml water.
9. Put 200μl into 96 well plate.
10. Air dry.
11. Add water 200μl.
12. Wait 20 min.
13. Remove water.
14. Add water 200μl.
15. Test OD 500
Biofilm Assay
Materials:
Equipment:
1. Pipette and pipette tips
2. Centrifuge
3. ELISA reader
4. Incubator
5. 96 well plate
6. Centrifuge tube
Consumables:
1. IPTG
2. LB
3. ddH2O
Protocol:
1. Culture bacteria NOS-DA-BL21, BW25113 csgD, BW25113 control for 2hr.
2. Add IPTG for induce for 12hr.
3. Adjust OD to 1.
4. Add congo red 3μl to 1ml bacteria.
5. Wait 2hr.
6. Centrifuge full speed 2min.
7. Remove supernatant.
NOS Kinetic Test
Materials:
Equipment:
1. Pipette and pipette tips
2. Centrifuge
3. ELISA reader
4. Incubator
5. 96 well plate
6. Centrifuge tube
Consumables:
1. NOS kit
2. IPTG
3. LB
4. ddH2O
Protocol:
Preparation:
1. Culture bacteria (NOSDA BL21) 3ml for 13 hr.
2. Adjust OD to 1.
3. Add IPTG 0.1 one hr.
4. Sonication.
5. Put 60μl of cell (NOSDA BL21) into 96well.
NOS assay:
1. Prepare 40μl reaction mix for each well.
| Components | Volume (μl) |
|---|---|
| NOS cofactor 1 | 10 |
| NOS cofactor 2 | 20 |
| Substrate | 5 |
| Nitrate reductase | 5 |
| Total Volume | 40 |
2. Add 40μl reaction mix for each well by 0, 5, 10, 15, 20, 30, 40, 50 min.
3. Incubate 10 mins 37oC.
4. Add 95μl NOS assay buffer to each well.
5. Add 5μl enhancer to each well.
6. Incubate room temperature 10mins.
7. Add 50μL reagent 1 and 50μL reagent 2 to each well.
8. Incubate for 10 mins.
9. Measure OD540.
IPTG Induce NOS Test
Materials:
Equipment:
1. Pipette and pipette tips
2. Pipette and pipette tips
3. Centrifuge
4. ELISA reader
5. Incubator
6. 96 well plate
7. Centrifuge tube
Consumables:
1. NOS kit
2. IPTG
3. LB
4. ddH2O
Protocol:
Preparation:
1. Culture bacteria (NOSDA BL21) 3ml for 12 hr.
2. Adjust OD to 0.5.
3. Add IPTG with different time (0.025mM, 0.05mM, 0.1mM) and concentration (0.5hr, 1hr, 1.5hr).
4. Sonication.
NOS assay kit:
1. Put 60μl of cell (NOSDA BL21) into 96well.
2. Prepare 40μl reaction mix for each well.
| Components | Volume (μl) |
|---|---|
| NOS cofactor 1 | 10 |
| NOS cofactor 2 | 20 |
| Substrate | 5 |
| Nitrate reductase | 5 |
| Total Volume | 40 |
3. Add 40μl reaction mix for each well by 0, 5, 10, 15, 20, 30, 40, 50 min.
4. Incubate 10 mins 37oC.
5. Add 95μl NOS assay buffer to each well.
6. Add 5μl enhancer to each well.
7. Incubate room temperature 10mins.
8. Add 50μL reagent 1 and 50μL reagent 2 to each well.
9. Incubate for 10 mins.
10. Measure OD540.
LB Preparation
Materials:
Equipment:
1. Glass bottle (for LB broth) or Erlenmeyer flask (for agar medium)
2. Magnetic stirrer
3. Magnetic stirring bar
4. Plastic measure jug
5. Measure cylinder
6. Spoon
7. Petri dish (for agar medium)
8. Aluminum foil
9. Electronic balance
10. Weighing paper or weighing bowl
11. Pipette and pipette tips
Consumables:
1. DW (distilled water)
2. Tryptone
3. Yeast extract
4. NaCl
5. NaOH (10N)
6. Agar (for LB agar)
7. Antibiotics (for selection plate)
Protocol:
1. Prepare mixture as the following inside a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.
| Components | Amount |
|---|---|
| DW | 98 ml |
| Tryptone | 1 g |
| Yeast Extract | 0.5 g |
| NaCl | 1 g |
| NaOH (10N) | 20 μl |
| Total Volume | 100 ml |
| Agar (for LB agar) | 1.50% |
2. Mix well, pour the mixture into the bottle or flask and autoclave. Notes: For agar medium, pour the mixture to the petri dish and dry the plates.
For selection plates, add antibiotics as the following before pouring the mixture to the petri dish:
Competent Cell Preparation
Protocol:
Equipment:
1. Glass bottle (for LB broth) or Erlenmeyer flask (for agar medium)
2. Magnetic stirrer
3. Magnetic stirring bar
4. Plastic measure jug
5. Measure cylinder
6. Spoon
7. Petri dish (for agar medium)
8. Aluminum foil
9. Electronic balance
10. Weighing paper or weighing bowl
11. Pipette and pipette tips
Consumables:
1. CaCl2
2. Glycerol
3. LB
4. ddH2O
Culture bacteria for making competent cell:
1. Add 6mL LB into the test tube.
2. Pick one colony from the plate and add it into the test tube.
3. Culture O.N. 37C, 200 rpm.
Test O.D. value & make competent cell:
1. Add 1mL overnight bacterial culture to 50mL LB.
2. When the O.D. is about 0.3~0.4, centrifuge 5000 rpm, 10 mins, discard the supernatant.
3. Resuspend the bacteria pellet in 25 ml ice cold 100 mM CaCl2 solution for 30 minutes on ice. Centrifuge the cell suspension at 5000 rpm in the Sorvall GSA rotor for 10 minutes. Discard the supernatant.
4. Resuspend the bacteria pellet on ice in 1ml of ice cold 100 mM CaCl2/15% glycerol (sterile) in each.
5. Dispense the bacteria in 100 mL aliquots (on ice) to 10 eppendorf.
6. Freeze cells at -80oC.
Standard PCR
Materials:
Equipment:
1. PCR tubes
2. Ice bucket
3. Thermocycler
4. Pipette and pipette tips
Consumables:
1. MQ or ddH2O
2. 2x PCR buffer for KOD FX
3. dNTPs 2 mM
4. Forward and reverse primer (10 μM)
5. KOD FX polymerase
Protocol:
1. Prepare 40μl reaction mix for each well.
| Components | Volume (μl) |
|---|---|
| MQ or ddH2O | 1.1 |
| 10x PCR buffer | 1 |
| dNTP 2.5 mM | 0.8 |
| Template | 1 |
| Forward primer 10 μM | 1 |
| Reverse primer 10 μM | 0.1 |
| Taq polymerase + vent | 0.1 |
| Total Volume | 10 |
2. Gently mix the PCR reactions and centrifuge briefly.
3. Transfer the PCR tubes to a thermocycler.
| step | Temperature | Time |
|---|---|---|
| Initial denaturalization | 94°C | 2 mins |
| 25 - 35 cycles | 98°C (denaturation) | 30 secs |
| 55°C (annealing) | 30 secs | |
| 68°C (extension) | 2 mins (depend on sequence size 2kbp/min.) | |
| Final Extension | 68°C | 2 mins |
| Hold | 16°C (holding for a short time) or 4°C (holding for a long time) | ∞ |
PCR Clean-up
Materials:
Equipment:
1. Eppendorf tubes
2. Collection tubes
3. Spin column
4. Pipette and pipette tips
5. Centrifuge
Consumables:
1. Isopropanol
2. Binding buffer
3. Washing buffer
4. Elution buffer
5. MQ or ddH2O
Protocol:
DNA binding:
1. Add MQ or ddH2O to DNA solution until the volume reaches 100 μl.
2. Add 300 μl of binding buffer to the solution.
3. Add 150 μl of isopropanol and invert the eppendorf 10 times.
4. Place a spin column in a provided collection tube. Transfer 550 μl sample to the spin column and centrifuge for 30 secs, 12,000 rpm.
5. Discard flow-through and place the spin column back into the collection tube.
Wash
1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 secs, 12,000 rpm.
2. Discard flow-through and place the spin column back into the collection tube.
3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 mins, 12,000 rpm.
4. Discard flow-through and place the spin column back into the collection tube.
DNA elution
1. Transfer spin column to clean eppendorf. Add 50 μl of elution buffer to the spin column. Centrifuge for 2 mins, 12,000 rpm.
2. Collect the pure sample in the eppendorf and discard the spin column.
3. Keep the DNA solution in -20°C freezer.
Restriction Enzyme Digestion
| Restriction Enzymes | Buffer (Buffer will depend on restriction enzyme being used) |
|---|---|
| EcoRI | SuR E/Cut Buffer H (10X) - Roche |
| XbaI | CutSmart Reaction Buffer (10x) - NEB or 3.1 - NEB |
| SpeI | CusSmart Reaction Buffer (10x) - NEB |
| PstI | 3.1 (10X) - NEB |
Materials:
Equipment:
1. Eppendorf tubes
2. Pipette and pipette tips
3. Ice bucket
4. Incubator (37°C)
Consumables:
1. Restriction enzymes
2. Buffer 10x (depends on its restriction enzymes)
3. DNA sample
4. MQ od ddH2O (for single digestion)
Protocol:
1. On the ice, add all the components.
Double Digestion:
| DNA | 15 μl |
| Buffer 10x | 3 μl |
| Restriction enzyme 1 | 1 μl |
| Restriction enzyme 2 | 1 μl |
| Total Volume | 20 μl |
|---|
Single Digestion (Structure Check):
| Plasmid DNA | 2 μl |
| Buffer 10x | 1.5 μl |
| Restriction enzyme | 0.5 μl |
| MQ or ddH2O | 11 μl |
| Total Volume | 15 μl |
|---|
2.Mix gently and incubate for 1-2 hours for double digestion or 30 mins - 1 hour for single digestion at 37°C. Note: Incubation time varies along the total volume of the reaction.
Gel Extraction and Purification
Materials:
Equipment:
1. Eppendorf tubes
2. Collection tubes
3. Spin column
4. Pipette and pipette tips
5. Centrifuge
6. Scalpel
7. Dry Thermounit
Consumables:
1. Isopropanol
2. Binding buffer
3. Washing buffer
4. Elution buffer
Protocol:
Gel excision, solubilization and DNA binding:
1. Excise band with scalpel and transfer to a new eppendorf tube.
2. Weigh the gel slice in a tube (by measuring the weight difference of an empty eppendorf and the eppendorf with gel slice in the tube).
3. Add 3 volumes of binding buffer to 1 volume of gel (100 mg = 100 μl).
4. Incubate at 60°C for 2-10 mins (or until the gel has completely dissolved).
5. Add 1.5 volume of isopropanol and invert the eppendorf 10 times.
6. Place a spin column in a provided collection tube. Transfer 700 μl sample to the spin column and centrifuge for 30 secs, 12,000 rpm.
7. Discard flow-through and place the spin column back into the collection tube.
2. Wash:
1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 secs, 12,000 rpm.
2. Discard flow-through and place the spin column back into the collection tube.
3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 mins, 12,000 rpm.
4. Discard flow-through and place the spin column back into the collection tube.
3. DNA elution:
1. Transfer spin column to clean eppendorf. Add 50 μl of elution buffer to the spin column. Centrifuge for 2 mins, 12,000 rpm.
2. Collect the pure sample in the eppendorf and discard the spin column.
Keep the DNA solution in -20°C freezer.
Ligation
Materials:
Equipment:
1. Eppendorf tubes
2. Pipette and pipette tips
3. Centrifuge
Consumables:
1. T4 DNA ligase (NEB)
2. T4 DNA ligase buffer (NEB)
Protocol:
1. Prepare 40μl reaction mix for each well.
| Components | Volume (μl) |
|---|---|
| Vector DNA | 12 |
| Insert DNA | 40 |
| Fast Ligase 2X buffer | 5.8 |
| T4 ligase | 0.5 |
| Total Volume | 58.3 |
2. Gently mix the reaction and centrifuge.
3. Incubate at 16°C overnight.
4. Keep the DNA solution in -20°C freezer.
Colony PCR
Materials:
Equipment:
1. PCR tubes
2. Ice
3. Thermocycler
4. Pipette and pipette tips
Consumables:
1. MQ or ddH2O
2. 10x PCR buffer
3. dNTPs 2.5 mM
4. Forward and reverse primer (10 μM)
5. Taq polymerase with 3 μl vent
Protocol:
1. On ice, add all components in a PCR tube, making up to 50 µl volume reaction.
| Components | Volume (μl) |
|---|---|
| MQ or ddH2O | 1.1 |
| 10x PCR buffer | 1 |
| dNTP 2.5 mM | 0.8 |
| Template | 1 |
| Forward primer 10 μM | 1 |
| Reverse primer 10 μM | 0.1 |
| Taq polymerase + vent | 0.1 |
| Total Volume | 10 |
2. Gently mix the PCR reactions and centrifuge briefly.
3. Transfer the PCR tubes to a thermocycler.
| step | Temperature | Time |
|---|---|---|
| Initial denaturalization | 94°C | 5 mins |
| 25 - 35 cycles | 94°C (denaturation) | 30 secs |
| 55°C (annealing) | 30 secs | |
| 72°C (Extension) | 2 mins (depend on sequence size 2kbp/min.) | |
| Final Extension | 72°C | 5 mins |
| Hold | 16°C (holding for a short time) or 4°C (holding for a long time) | ∞ |
Bacterial Glycerol Stock
Materials:
Equipment:
1. Pipette and pipette tips
2. Cryovial
Consumables:
1. 50% glycerol (autoclaved)
2. Bacterial overnight culture with an antibiotic if necessary
Protocol:
1. Put 1 ml of bacterial overnight culture into the cryovial and add 500 μl of 50% glycerol. Mix it well.
2. Keep it in the -80°C freezer.
SDS-PAGE
Materials:
Equipment:
1. Eppendorf tubes
2. Pipette and pipette tips
3. Centrifuge
4. Vortex
5. Ice bucket
6. Empty box
7. Dry Thermounit
8. Glass plates
9. 10-well comb
10. Spacer
11. Clamp
12. Casting stand
13. Buffer tank
14. Voltage source
15. Shaker
Consumables:
1. H2O
2. 30% acrylamide
3. 1.5M tris pH 8.8
4. 1M tris pH 6.8
5. 10% SDS
6. 10% APS
7. TEMED
8. Prestained Protein Marker - Bioman
9. Protein dye
10. Overnight culture (sample)
11. Coomassie Brilliant Blue (CBB) staining solution
12. Destaining buffer
13. Tank buffer (1X)
14. Isopropanol
Protocol:
Resolving gel preparation:
1. Adjust the amount of agarose to get the desired gel concentration.
2. In a 50 ml eppendorf tube add all the components 15% gel concentration.
| Total Volume | 5 ml | 10 ml | 15 ml |
|---|---|---|---|
| H2O (ml) | 1.15 | 2.3 | 3.4 |
| 30% acrylamide mix (ml) | 2.5 | 5 | 7.5 |
| 1.5M Tris pH 8.8 (ml) | 1.25 | 2.5 | 3.3 |
| 10% SDS (ml) | 0.05 | 0.1 | 0.15 |
| 10% APS (ml) | 0.1 | 0.2 | 0.3 |
| TEMED (ml) | 0.002 | 0.004 | 0.006 |
3. Pour the mixture in between the glasses and add iso-propanol afterward.
4. Wait for 10-15 mins or until the gel solidifies.
5. Pour out the iso-propanol.
Stacking gel preparation:
1. In a 50 ml eppendorf tube add all the components.
| Total Volume | 1.5 ml | 3 ml | 5 ml |
|---|---|---|---|
| H2O (ml) | 1.05 | 2.1 | 3.4 |
| 30% acrylamide mix (ml) | 0.25 | 0.5 | 0.83 |
| 1M Tris pH 6.8 (ml) | 0.19 | 0.38 | 0.63 |
| 10% SDS (ml) | 0.015 | 0.03 | 0.05 |
| 10% APS (ml) | 0.03 | 0.06 | 0.05 |
| TEMED (ml) | 0.0015 | 0.003 | 0.005 |
2. Pour the mixture in between the glass plates and add the 10-well comb.
3. Wait for 10-15 mins or until the gel solidifies.
Sample preparation:
1. Take overnight cultures with the desired volume and centrifuge.
2. Take 12 μl of pellet or supernatant (depends on necessity) and move to a new fresh eppendorf.
3. Add 3 μl dye into the eppendorf. Vortex and centrifuge briefly.
4. Heat the eppendorf at 100°C for 10 mins and centrifuge briefly.
5. Put samples in the ice bucket.
Gel running:
1. When the gel solidified, set up the running equipment and pour 1X tank buffer.
2. Take out the comb and load 3 μl of the marker into a well and load the samples into the remaining wells.
3. Run the first 30 mins at 120V then continue to run for 1 hour at 150 V. Note: voltage and time may vary.
Staining:
1. Take out the glass plates out of the buffer tank and split up the glass plates to take out the gel.
2. The stacking gel and put the resolving gel inside an empty box, then add the CBB staining solution until it covers the gel.
3. Put the box on a shaker and shake for 30 mins or until the gel turns blue.
Destaining:
1. Pour the CBB staining solution back into the bottle and add the destaining buffer into the box until it covers the gel.
2. Put the box on a shaker and shake until the protein bands are visible or until the gel turns white.
3. Pour out the destaining buffer.
