Revision as of 09:34, 17 October 2019

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Collab

Protocols

This page is about our protocols, you can find all the experimental process you need.
Enjoy!

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 1mL of bacterial overnight culture into the cryovial and add 500uL of 50% glycerol. Mix it well.

2. Keep it in the -80°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 2.5mM

4. Forward and reverse primer (10uM)

5. Taq polymerase with 3ul vent

Protocol:

1. On ice, add all components in a PCR tube, making up to 50 µl volume reaction.

Components	Volume(ul)
MQ or ddH₂O	1.1
10x PCR buffer	1
dNTP 2.5mM	0.8
Template	1
Forward primer 10uM	1
Reverse primer 10uM	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)	∞

Electroporation Escherichia coli for homologous

Materials:

Equipment:

1. Pipette and pipette tips

2. Eppendorf tubes

3. Centrifuge

4. Flasks

5. Electroporation cuvettes (2mm gap)

6. Electroporator

Consumables:

1. SOB

2. Chilled 10% glycerol

3. Soc

4. Selection plates

5. DNA fragment

6. 10% L-arabinose

7. Ampicillin

Protocol:

Competent cell making:

1. Inoculate 500ul of overnight culture (Escherichia coli with pKD46) to 50ml of fresh SOB-Mg medium with 50ul of ampicillin at 30°C.

2. Grow cells with shaking at 30°C for 1 hour and 50 mins.

3. Add 1ml of 10% L-arabinose and culture for 1 hour.

4. Transfer the culture into 50ml centrifuge tube, and centrifuge it for 5 min at 4°C and 6,000 rpm to harvest cells. (After this stage, the cells should be kept cool throughout the cell preparation)

5. Remove the supernatant and suspend the cells with 1ml of chilled 10% glycerol. Transfer the cell suspension into a 1.5ml tube and centrifuge again for 1 min at 4°C and 12,000rpm. (first wash)

6. Remove the supernatant using a pipette and suspend the cells with 1ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm. (second wash)

7. Remove the supernatant using a pipette and suspend the cells with 1ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm. (third wash)

8. Remove the supernatant using a pipette and suspend the cells with 250ul (depends on the amount of the cell) of chilled 10% glycerol.

Electroporation:

1. Mix 50ul of the competent cell and 5ul DNA solution in a fresh 1.5ml tube.

2. Transfer the mixture into chilled electroporation cuvette (2mm gap), and pulse (Ec 2).

3. Add 1ml of SOC with 20ul 10% arabinose immediately after the pulse.

4. Incubate it for 1 hour at 37°C.

5. Plate 100ul of the culture on a selection plate, and incubate at 30°C.

Electroporation Escherichia coli with plasmid

Materials:

Equipment:

1. Pipette and pipette tips

2. Eppendorf tubes

3. Centrifuge

4. Flasks

5. Electroporation cuvettes (2mm gap)

6. Eletroporator

Consumables:

1. SOB

2. Chilled 10% glycerol

3. Soc

4. Selection plates

5. DNA plasmid

Protocol:

Competent cell making:

1. Inoculate 1ml of overnight culture to 50ml of fresh SOB-Mg medium.

2. Grow cells with shaking at 37°C for 1.5 hours (recA+ strain) or 1.75 hours (recA- strain).

3. Chill the flask on ice for 5 min, transfer the culture into 50ml centrifuge tube, and centrifuge it for 5 min at 4°C and 6,000 rpm to harvest cells. (After this stage, the cells should be kept cool throughout the cell preparation)

4. Remove the supernatant and suspend the cells with 1ml of chilled 10% glycerol. Transfer the cell suspension into a 1.5ml tube and centrifuge again for 1 min at 4°C and 12,000rpm.

5. Remove the supernatant using a pipette and suspend the cells with 1ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm.

6. Remove the supernatant using a pipette and suspend the cells with 250ul (depends on the amount of the cell) of chilled 10% glycerol.

Electroporation:

1. Mix 50ul of the competent cell and DNA solution (the amount depends on the DNA plasmid) in a fresh 1.5ml tube.

2. Transfer the mixture into chilled electroporation cuvette (2mm gap), and pulse (Ec 2).

3. Add 1ml of SOC immediately after the pulse.

4. Incubate it for 1 hour at 37°C.

5. Plate the culture on a selection plate, and incubate.

Gel Electrophoresis

Materials:

Equipment:

1. Microwave

2. Electrophoresis comb

3. Electrophoresis trays

4. Pipette and pipette tips

5. Buffer tank

6. Voltage source

7. Glass bottle or Erlenmeyer flask

Consumables:

1. Agarose powder

2. TBE or TAE buffer

3. λ EH (λ DNA/EcoRI+HindIII) marker (2X) - Promega

4. 2X SLB or 10X SLB (sample loading buffer)

5. DNA sample

6. EtBr (Ethidium bromide)

7. RO (reverse osmosis)

Protocol:

Agarose gel preparation:

1. Adjust the amount of agarose to get the desired gel concentration (in this protocol, we’ll be using 0.7% agarose).

2. Weight 0.7 grams of agarose powder and add it to a glass bottle or Erlenmeyer flask.

3. Measure 100mL of TBE buffer or TAE buffer and add it to the glass bottle or Erlenmeyer.

4. Put the mix into the microwave and heat it until the agarose is completely dissolved.

5. Let the mix cool down and pour it to the electrophoresis tray.

6. Set the electrophoresis comb and wait for the gel to solidify.

Sample preparation:

1. Samples are prepared in various ways depends on its proposes.

Purpose	DNA Solution	SLB	Total Volume
Size checking	2uL	10uL (2X)	12uL
Gel extraction	depends on how much DNA solution to be extracted	5uL (10X)

Agarose gel running:

1. When the agarose gel solidified, take out the electrophoresis comb.

2. Pour the TBE buffer or TAE buffer to the buffer tank. Note: buffer used for running has to be the same as the buffer used to prepare the gel solution.

3. Put the gel in the buffer tank.

4. Load 5uL λ EH (λ DNA/EcoRI+HindIII) marker (2X) in a well and load the DNA sample mix with SLB in the remaining wells.

5. Run for 20 mins at 100V. Note: voltage and time varies.

Agarose gel visualization:

1. Take the gel out of the buffer tank.

2. Soak the gel in the EtBr solution for 8 mins and in RO for 30 secs.

3. Take the gel out of RO and visualize the DNA under UV transilluminator.

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 (100mg = 100ul).

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 700ul 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 500ul of washing buffer to the spin column and centrifuge for 30 secs, 12,000rpm.

2. Discard flow-through and place the spin column back into the collection tube.

3. Add 200ul of washing buffer to the spin column and centrifuge for 5 mins, 12,000rpm.

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 50ul of elution buffer to the spin column. Centrifuge for 2 mins, 12,000rpm.

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. Set up the following reaction on ice:

Total Volume	58.3uL
Vector	12uL
Insert	40uL
T4 DNA ligase buffer (10x)	5.8uL
T4 DNA ligase	0.5uL

2. Gently mix the reaction and centrifuge.

3. Incubate at 16°C overnight.

4. Keep the DNA solution in -20°C freezer.

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 100uL.

2. Add 300uL of binding buffer to the solution.

3. Add 150uL of isopropanol and invert the eppendorf 10 times.

4. Place a spin column in a provided collection tube. Transfer 550uL 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 500uL of washing buffer to the spin column and centrifuge for 30 secs, 12,000rpm.

2. Discard flow-through and place the spin column back into the collection tube.

3. Add 200uL of washing buffer to the spin column and centrifuge for 5 mins, 12,000rpm.

4. Discard flow-through and place the spin column back into the collection tube.

DNA elution

1. Transfer spin column to clean eppendorf. Add 50uL of elution buffer to the spin column. Centrifuge for 2 mins, 12,000rpm.

2. Collect the pure sample in the eppendorf and discard the spin column.

3. Keep the DNA solution in -20°C freezer.

Plasmid Isolation: Miniprep

Materials:

Equipment:

1. Eppendorf tubes

2. Collection tubes

3. Spin column

4. Pipette and pipette tips

5. Centrifuge

6. Ice bucket

Consumables:

1. Suspension buffer

2. Lysis buffer

3. Binding buffer

4. Washing buffer I

5. Washing buffer II

6. Elution buffer

7. LB broth

8. Antibiotic as needed

Protocol:

1. Prepare 2-5ml overnight culture in LB medium with the antibiotic (depends on the plasmid to be extracted).

2. Collect cells from the overnight culture with a centrifuge (12,000rpm 30sec) and discard the supernatant.

3. Add 250ul of suspension buffer and suspend bacterial cells by vortex.

4. Add 250ul of lysis buffer and mix gently by inverting the tube 10 times.

5. Add 350ul of binding buffer and mix gently by inverting the tube 10 times.

6. Incubate on ice for 5 mins.

7. Centrifuge at 4°C, 12,000rpm for 10 mins.

8. Place a spin column in a provided collection tube. Transfer 750ul of supernatant to spin column.

9. Centrifuge at room temperature, 12,000 rpm for 30 sec and discard the flow-through.

10. Add 500ul of washing buffer I to the spin column and centrifuge for 30 sec. Discard the flow-through.

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:

Total Volume	50uL
DNA	43uL
Buffer 10x	5uL
Restriction enzyme 1	1uL
Restriction enzyme 2	1uL

Single Digestion(Structure Check):

Total Volume	15uL
Plasmid DNA	2uL
Buffer 10x	1.5uL
Restriction enzyme	0.5uL
MQ or ddH₂O	11uL

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.

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. CBB (Coomassie Brilliant Blue) staining solution

12. Destaining buffer

13. Tank buffer (1X)

14. Iso-propanol

Protocol:

Resolving gel preparation

1. Adjust the amount of agarose to get the desired gel concentration

2. In a 50ml eppendorf tube add all the components 15% gel concentration

Total Volume	5mL	10mL	15mL
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)	0.10	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 50mL eppendorf tube add all the components

Total Volume	1.5ml	3ml	5ml
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 12uL of pellet or supernatant (depends on necessity) and move to a new fresh eppendorf.

3. Add 3uL 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 3uL 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 150V. 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.

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 to 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.

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 2mM

4. Forward and reverse primer (10uM)

5. KOD FX polymerase

Protocol:

1. On the ice, add all components in a PCR tube, making up to 50 µl volume reaction.

Components	Volume(ul)
MQ or ddH₂O	1.1
10x PCR buffer	1
dNTP 2.5mM	0.8
Template	1
Forward primer 10uM	1
Reverse primer 10uM	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	Temp	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
Final Extension	68°C	2 mins
Hold	16°C (holding for a short time) or 4°C (holding for a long time)	∞

Media Preparation

1.LB (Lysogeny Broth)

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.

Total Volume	100mL
DW	98mL
Tryptone	1g
Yeast Extract	0.5g
NaCl	1g
NaOH (10N)	20uL
Agar (for LB agar)	1.5%

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:

Kanamycin: 100uL/100ml media

Chloramphenicol: 50uL/100mL media

Ampicillin: 100uL/100mL media

2.SOB (Super Optimal Broth)

Materials:

Equipment:

1. Flask

2. Magnetic stirrer

3. Magnetic stirring bar

4. Plastic measure jug

5. Measure cylinder

6. Spoon

7. Aluminum foil

8. Electronic balance

9. Weighing paper or weighing bowl

10. Pipette and pipette tips

Consumables:

1. DW (distilled water)

2. Tryptone

3. Yeast extract

4. 5M NaCl

5. 3M KCL

Protocol:

1. Prepare mixture as the following inside a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.

Total Volume	100ml
DW	96ml
Tryptone	2g
Yeast Extract	0.5g
5M NaCl	0.2ml
3M KCL	83uL

2. Mix well, pour the mixture into the flask and autoclave.

3.SOC

Materials:

Equipment:

1. Pipette and pipette tips

Consumables:

1. SOB medium in a glass bottle

2. 1M M_gCl₂

3. 1M M_gSO₄

4. 20% glucose

Protocol:

1. Add chemicals as the following to a bottle of 100ml SOB (autoclaved)

1M M_gCl₂	96 ml
1M M_gSO₄	2g
20% glucose	0.5g

4. M9 Medium

Materials:

Equipment:

1. Pipette and pipette tips

Consumables:

1. M9 salts(5x)

2. 20% glucose

3. 1M M_gSO₄

4. 1M C_aCl₂

5. H₂O

Protocol:

1. Add chemicals as the following to a bottle

Total Volume	100ml
M9 salts (5X)	20ml
20% glucose	2ml
1M M_gSO₄	200uL
1M C_aCl₂	10uL
H₂O	78ml

2. Mix well, pour the mixture into the flask and autoclave.

Functional Test

can gene kill switch functional test

Materials:

Equipment:

1. CO₂ incubator

2. Toothpick

Consumables:

1. LB plates (can also with the desired antibiotic)

Protocol:

1. Divide the plate into several parts depends on how many colonies are being tested.

2. Streak the plates using the toothpick.

3. Grow the bacteria in the 37°C CO₂ incubator and normal incubator for comparison.

dap gene kill switch functional test

Materials:

Equipment:

1. 37°C incubator

2. Toothpick

Consumables:

1. LB plates (can also with the desired antibiotic)

2. LB + dap plates (concentration of dap may vary)

Protocol:

1. Divide the plates with dap chemical and without dap chemical into several parts depends on how many colonies are being tested.

2. Streak the plates using the toothpick.

3. Grow the bacteria in the 37°C incubator.

p-Cresol Sensing Functional Test

Materials:

Equipment:

1. 96 well-plate

2. Pipette and tips

3. 37°C incubator

4. Spectrofluorometer

Consumables:

1. Bacterial culture

2. p-Cresol

Protocol:

1. Make overnight cultures induced with different concentrations of p-Cresol and also without p-Cresol for control. Incubate at 37°C incubator.

2. Load 200ul of samples onto the 96 well plates. Arrange with your personal preference.

3. Measure fluorescence using the spectrofluorometer.

Spot on Lawn Assay

Materials:

Equipment:

1. Pipette and tips

2. -80°C fridge

3. 37°C anaerobic incubator

Consumables:

1. Bacterial culture

2. BHI plate

3. 0.22um filter

4. Purified bacteriocin (positive control)

5. LB medium

Protocol:

1. Grow overnight cultures of c. difficile and bacteria carrying plasmid with bacteriocin in 6ml LB.(Note: In this experiment, total cell lysate and filtered growth medium of the culture carrying plasmid with bacteriocin will be used to test both the expression and secretion of bacteriocin.)

Take 100ul of the c. difficile overnight culture and streak evenly on the dried BHI plate.

To test the secretion ability of bacteriocin, take 2ml overnight culture of bacteriocin producing bacteria and filter it through a 0.22um filter. Spot 5 ul of the filtered solution on the streaked BHI plate.

To test the expression of bacteriocin, take 2ml overnight culture of bacteriocin producing bacteria and put it in -80°C fridge. Freeze for 10 min and take the frozen bacteria back to the room temperature to unfroze it. Repeat this step twice. Then, take the culture solution and filter it through a 0.22um filter and spot 5ul of the filtered solution on the streaked BHI plate.

For positive control, purified bacteriocin without yebF secretion tag kindly proved by one of the PI’s lab are used.

Grow the plate in the anaerobic incubator and observe the inhibition zone.

TAL Functional Test

Materials:

Equipment:

1. 37°C anaerobic incubator

2. Pipette and tips

3. Spectrophotometer （NanoDrop）

Consumables:

Bacterial culture

1. n-octanol

2. LB

3. LB + 2mM tyrosine

4. LB + 1mM tyrosine

5. LB + 500uM tyrosine

6. p-Coumaric acid

7. Lysis buffer

8. Neutralization buffer

Protocol:

p-Coumaric acid fermentation:

1. For each E.coli strain, bacterial colony is inoculate to 6ml LB medium containing appropriate antibiotic and grow overnight in normal 37°C incubator.

2. Incubate for 16 hours.

3. Take 60ul of bacterial culture to refresh in 6ml LB containing containing appropriate antibiotic and different concentration of tyrosine.

4. Incubate the refreshed cell culture in anaerobic incubator at 37°C.

5. Samples are collected in the 24- and 48- hour, after the initiation of the anaerobic 37°C incubation.

Sample collection:

1. 250uL of samples are collected at the 2 particular times stated above.

2. The positive control is prepared by collecting 225ul of negative control(Wild-type strain) culture with 25ul of 500uM, 250uM and 50uM of p-Coumaric acid in LB to create a bacterial culture with p-Coumaric acid concentration of 50uM, 25uM and 5uM.

Sample Extraction:

1. Directly after sample collection, the samples are lysed with 25ul of Lysis buffer(PD2) and neutralized with 43.5ul neutralization buffer(PD3). (Thermo plasmid MiniPrep)

2. All samples are acidified in 12.5ul concentrated acetic acid and vortexed.

3. Samples are then induced with 50ul n-octanol (the organic phase in two-phase extraction).

4. Vortexing and centrifugation ( >12000g, 1 min ) followed and the upper organic phase is collected for measurement.

Measurement:

1. The absorbance in the UV-spectrum (190nm-350nm) of the octanol extracts is measured in a spectrophotometer (NanoDrop).

2. The spectrophotometer blanked by pure n-octanol.

3. All measurements are done on 2ul of extractant.

4. Absorbance measurements are done in the same way on the standards of p-Coumaric acid in octanol prepared from solid p-Coumaric acid.

@@ Line 1,354: / Line 1,354: @@
                           <p>1. Divide the plate into several parts depends on how many colonies are being tested.</p>
                           <p>2. Streak the plates using the toothpick.</p>
-                          <p>3. Grow the bacteria in the 37 °C CO<sub>2</sub> incubator and normal incubator for comparison.</p>
+                          <p>3. Grow the bacteria in the 37°C CO<sub>2</sub> incubator and normal incubator for comparison.</p>
                        </div>
                      </div>
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                    </div>
                    <div class="container margin-top-lg">
-                     <div class="align-font bold-font"><h3 style="font-size: 2rem;">p-Cresol Sensing Functional Test</h3></div>
+                     <div class="align-font bold-font"><h3 style="font-size: 2rem;"><i>p</i>-Cresol Sensing Functional Test</h3></div>
                      <hr class="half-hr margin-bottom">
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                        <div class="col">
-                         <br><br><div style="font-size: 2rem; text-align: center;margin-bottom: 1rem;">Materials:</div>
+                         <br><br><div style="font-size: 2rem; text-align: center;margin-bottom: 1rem; font-weight: bold;">Materials:</div>
                          <p><h4>Equipment:</h4></p>
-                         <ol>
+                         <p>1. 96 well-plate</p>
-                          <li><p>96 well-plate</p></li>
+                        <p>2. Pipette and tips</p>
-                          <li><p>Pipette and tips</p></li>
+                        <p>3. 37°C incubator</p>
-                          <li><p>37 °C incubator</p></li>
+                        <p>4. Spectrofluorometer</p>
-                          <li><p>Spectrofluorometer</p></li>
-                        </ol>
                          <span style="font-size: 1rem;font-weight: bold;"><h4>Consumables:</h4></span>
-                         <ol>
+                         <p>1. Bacterial culture</p>
-                          <li><p>Bacterial culture</p></li>
+                        <p>2. <i>p</i>-Cresol</p>
-                          <li><p>p-Cresol</p></li>
-                        </ol>
                        </div>
                        <div class="col">
-                         <div style="font-size: 2rem; text-align: center;margin-bottom: 1rem;">Method by step:</div>
+                         <br><br><div style="font-size: 2rem; text-align: center;margin-bottom: 1rem; font-weight:bold;">Protocol:</div>
-                         <ol>
+                         <p>1. Make overnight cultures induced with different concentrations of <i>p</i>-Cresol and also without p-Cresol for control. Incubate at 37°C incubator.</p>
-                          <li><p>Make overnight cultures induced with different concentrations of p-Cresol and also without p-Cresol for control. Incubate at 37 °C incubator.</p></li>
+                        <p>2. Load 200ul of samples onto the 96 well plates. Arrange with your personal preference.</p>
-                          <li><p>Load 200ul of samples onto the 96 well plates. Arrange with your personal preference.</p></li>
+                        <p>3. Measure fluorescence using the spectrofluorometer.</p>
-                          <li><p>Measure fluorescence using the spectrofluorometer.</p></li>
-                        </ol>
                        </div>
                      </div>
                    </div>
                    <div class="container margin-top-lg">
-                     <div class="align-font bold-font"><h3>Spot on Lawn Assay</h3></div>
+                     <div class="align-font bold-font"><h3 style="font-size: 2rem;">Spot on Lawn Assay</h3></div>
                      <hr class="half-hr margin-bottom">
                      <div class="row">
                        <div class="col">
-                         <div style="font-size: 2rem; text-align: center;margin-bottom: 1rem;">Materials:</div>
+                         <br><br><div style="font-size: 2rem; text-align: center;margin-bottom: 1rem; font-weight: bold;">Materials:</div>
-                         <h4>Equipment:</h4>
+                         <p><h4>Equipment:</h4></p>
-                        <ol>
+                        <p>1. Pipette and tips</p>
-                          <li><p>Pipette and tips</p></li>
+                        <p>2. -80°C fridge</p>
-                          <li><p>-80°C fridge</p></li>
+                        <p>3. 37°C anaerobic incubator</p>
-                          <li><p>37 °C anaerobic incubator</p></li>
-                        </ol>
                          <span style="font-size: 1rem;font-weight: bold;"><h4>Consumables:</h4></span>
-                         <ol>
+                         <p>1. Bacterial culture</p>
-                          <li><p>Bacterial culture</p></li>
+                        <p>2. BHI plate</p>
-                          <li><p>BHI plate</p></li>
+                        <p>3. 0.22um filter</p>
-                          <li><p>0.22um filter</p></li>
+                        <p>4. Purified bacteriocin (positive control)</p>
-                          <li><p>Purified bacteriocin (positive control)</p></li>
+                        <p>5. LB medium</p>
-                          <li><p>LB medium</p></li>
-                        </ol>
                        </div>
                        <div class="col">
-                         <div style="font-size: 2rem; text-align: center;margin-bottom: 1rem;">Method by step:</div>
+                         <br><br><div style="font-size: 2rem; text-align: center;margin-bottom: 1rem;">Protocol:</div>
-                         <ol>
+                         <p>1. Grow overnight cultures of <i>c. difficile</i> and bacteria carrying plasmid with bacteriocin in 6ml LB.<class="small-font bold-font">(Note: In this experiment, total cell lysate and filtered growth medium of the culture carrying plasmid with bacteriocin will be used to test both the expression and secretion of bacteriocin.)</p>
-                          <li><p>Grow overnight cultures of c. difficile and bacteria carrying plasmid with bacteriocin in 6ml LB.</p></li>
+                        <p>Take 100ul of the <i>c. difficile</i> overnight culture and streak evenly on the dried BHI plate. </p>
-                          <p class="small-font bold-font">&emsp;（Note: In this experiment, total cell lysate and filtered growth medium of the culture carrying plasmid with bacteriocin will be used to test both the expression and secretion of bacteriocin.）</p>
+                        <p>To test the secretion ability of bacteriocin, take 2ml overnight culture of bacteriocin producing bacteria and filter it through a 0.22um filter. Spot 5 ul of the filtered solution on the streaked BHI plate.</p>
-                          <li><p>Take 100ul of the c. difficile overnight culture and streak evenly on the dried BHI plate. </p></li>
+                        <p>To test the expression of bacteriocin, take 2ml overnight culture of bacteriocin producing bacteria and put it in -80°C fridge. Freeze for 10 min and take the frozen bacteria back to the room temperature to unfroze it. Repeat this step twice. Then, take the culture solution and filter it through a 0.22um filter and spot 5ul of the filtered solution on the streaked BHI plate.</p>
-                          <li><p>To test the secretion ability of bacteriocin, take 2ml overnight culture of bacteriocin producing bacteria and filter it through a 0.22um filter. Spot 5 ul of the filtered solution on the streaked BHI plate.</p></li>
+                        <p>For positive control, purified bacteriocin without yebF secretion tag kindly proved by one of the PI’s lab are used.</p>
-                          <li><p>To test the expression of bacteriocin, take 2ml overnight culture of bacteriocin producing bacteria and put it in -80°C fridge. Freeze for 10 min and take the frozen bacteria back to the room temperature to unfroze it. Repeat this step twice. Then, take the culture solution and filter it through a 0.22um filter and spot 5ul of the filtered solution on the streaked BHI plate.</p></li>
+                        <p>Grow the plate in the anaerobic incubator and observe the inhibition zone.</p>
-                          <li><p>For positive control, purified bacteriocin without yebF secretion tag kindly proved by one of the PI’s lab are used.</p></li>
-                          <li><p>Grow the plate in the anaerobic incubator and observe the inhibition zone.</p></li>
-                        </ol>
                        </div>
                      </div>
                    </div>
                    <div class="container margin-top-lg">
-                     <div class="align-font bold-font"><h3>TAL Functional Test</h3></div>
+                     <div class="align-font bold-font"><h3 style="font-size: 2rem;">TAL Functional Test</h3></div>
                      <hr class="half-hr margin-bottom">
                      <div class="row">
                        <div class="col">
-                         <div style="font-size: 2rem; text-align: center;margin-bottom: 1rem;">Materials:</div>
+                         <br><br><div style="font-size: 2rem; text-align: center;margin-bottom: 1rem; font-weight: bold;">Materials:</div>
-                         <h4>Equipment:</h4>
+                         <p><h4>Equipment:</h4></p>
-                        <ol>
+                        <p>1. 37°C anaerobic incubator</p>
-                          <li><p>370C anaerobic incubator</p></li>
+                        <p>2. Pipette and tips</p>
-                          <li><p>Pipette and tips</p></li>
+                        <p>3. Spectrophotometer <span class="small-font">（NanoDrop）</span></p>
-                          <li><p>Spectrophotometer <span class="small-font">（NanoDrop）</span></p></li>
-                        </ol>
                          <span style="font-size: 1rem;font-weight: bold;"><h4>Consumables:</h4></span>
-                         <ol>
+                         <p>Bacterial culture</p>
-                          <li><p>Bacterial culture</p></li>
+                        <p>1. <i>n</i>-octanol</p>
-                          <li><p>n-octanol</p></li>
+                        <p>2. LB</p>
-                          <li><p>LB</p></li>
+                        <p>3. LB + 2mM tyrosine</p>
-                          <li><p>LB + 2mM tyrosine</p></li>
+                        <p>4. LB + 1mM tyrosine</p>
-                          <li><p>LB + 1mM tyrosine</p></li>
+                        <p>5. LB + 500uM tyrosine</p>
-                          <li><p>LB + 500uM tyrosine</p></li>
+                        <p>6. <i>p</i>-Coumaric acid</p>
-                          <li><p>p-Coumaric acid</p></li>
+                        <p>7. Lysis buffer</p>
-                          <li><p>Lysis buffer</p></li>
+                        <p>8. Neutralization buffer</p>
-                          <li><p>Neutralization buffer</p></li>
-                        </ol>
                        </div>
                        <div class="col">
-                         <div style="font-size: 2rem; text-align: center;margin-bottom: 1rem;">Method by step:</div>
+                         <br><br><div style="font-size: 2rem; text-align: center;margin-bottom: 1rem;">Protocol:</div>
-                         <h5 class="bold-font">p-Coumaric acid fermentation:</h5>
+                         <h5 class="bold-font"><i>p</i>-Coumaric acid fermentation:</h5>
-                         <ol>
+                         <p>1. For each <i>E.coli</i> strain, bacterial colony is inoculate to 6ml LB medium containing appropriate antibiotic and grow overnight in normal 37°C incubator.</p>
-                          <li><p>For each E.coli strain, bacterial colony is inoculate to 6ml LB medium containing appropriate antibiotic and grow overnight in normal 370C incubator.</p></li>
+                        <p>2. Incubate for 16 hours. </p>
-                          <li><p>Incubate for 16 hours. </p></li>
+                        <p>3. Take 60ul of bacterial culture to refresh in 6ml LB containing containing appropriate antibiotic and different concentration of tyrosine.</p>
-                          <li><p>Take 60ul of bacterial culture to refresh in 6ml LB containing containing appropriate antibiotic and different concentration of tyrosine.</p></li>
+                        <p>4. Incubate the refreshed cell culture in anaerobic incubator at 37°C.</p>
-                          <li><p>Incubate the refreshed cell culture in anaerobic incubator at 370C.</p></li>
+                        <p>5. Samples are collected in the 24- and 48- hour, after the initiation of the anaerobic 37°C incubation.</p>
-                          <li><p>Samples are collected in the 24- and 48- hour, after the initiation of the anaerobic 370C incubation.</p></li>
-                        </ol>
                          <h5 class="bold-font">Sample collection:</h5>
-                         <ol>
+                         <p>1. 250uL of samples are collected at the 2 particular times stated above.</p>
-                          <li><p>250uL of samples are collected at the 2 particular times stated above. </p></li>
+                        <p>2. The positive control is prepared by collecting 225ul of negative control(Wild-type strain) culture with 25ul of 500uM, 250uM and 50uM of <i>p</i>-Coumaric acid in LB to create a bacterial culture with <i>p</i>-Coumaric acid concentration of 50uM, 25uM and 5uM.</p>
-                          <li><p>The positive control is prepared by collecting 225ul of negative control(WT strain) culture with 25ul of 500uM, 250uM and 50uM of p-Coumaric acid in LB to create a bacterial culture with p-Coumaric acid concentration of 50uM, 25uM and 5uM.</p></li>
-                        </ol>
                          <h5 class="bold-font">Sample Extraction:</h5>
-                         <ol>
+                         <p>1. Directly after sample collection, the samples are lysed with 25ul of Lysis buffer(PD2) and neutralized with 43.5ul neutralization buffer(PD3). (Thermo plasmid MiniPrep)</p>
-                          <li><p>Directly after sample collection, the samples are lysed with 25ul of Lysis buffer(PD2) and neutralized with 43.5ul neutralization buffer(PD3). (Thermo plasmid MiniPrep)</p></li>
+                        <p>2. All samples are acidified in 12.5ul concentrated acetic acid and vortexed.</p>
-                          <li><p>All samples are acidified in 12.5ul concentrated acetic acid and vortexed.</p></li>
+                        <p>3. Samples are then induced with 50ul n-octanol (the organic phase in two-phase extraction).</p>
-                          <li><p>Samples are then induced with 50ul n-octanol (the organic phase in two-phase extraction).</p></li>
+                        <p>4. Vortexing and centrifugation ( >12000g, 1 min ) followed and the upper organic phase is collected for measurement.</p>
-                          <li><p>Vortexing and centrifugation ( >12000g, 1 min ) followed and the upper organic phase is collected for measurement.</p></li>
-                        </ol>
                          <h5 class="bold-font">Measurement:</h5>
-                         <ol>
+                         <p>1. The absorbance in the UV-spectrum (190nm-350nm) of the octanol extracts is measured in a spectrophotometer (NanoDrop).</p>
-                          <li><p>The absorbance in the UV-spectrum (190nm-350nm) of the octanol extracts is measured in a spectrophotometer (NanoDrop).</p></li>
+                        <p>2. The spectrophotometer blanked by pure <i>n</i>-octanol.</p>
-                          <li><p>The spectrophotometer blanked by pure n-octanol.</p></li>
+                        <p>3. All measurements are done on 2ul of extractant.</p>
-                          <li><p>All measurements are done on 2ul of extractant.</p></li>
+                        <p>4. Absorbance measurements are done in the same way on the standards of <i>p</i>-Coumaric acid in octanol prepared from solid <i>p</i>-Coumaric acid.</p>
-                          <li><p>Absorbance measurements are done in the same way on the standards of p-Coumaric acid in octanol prepared from solid p-Coumaric acid.</p></li>
-                        </ol>
                        </div>
                      </div>

Difference between revisions of "Team:NCKU Tainan/Protocols"

Revision as of 09:34, 17 October 2019

Protocols

This page is about our protocols, you can find all the experimental process you need. Enjoy!

Bacterial Glycerol Stock

Equipment:

Consumables:

Colony PCR

Equipment:

Consumables:

Electroporation Escherichia coli for homologous

Equipment:

Consumables:

Competent cell making:

Electroporation:

Electroporation Escherichia coli with plasmid

Equipment:

Consumables:

Competent cell making:

Electroporation:

Gel Electrophoresis

Equipment:

Consumables:

Agarose gel preparation:

Sample preparation:

Agarose gel running:

Agarose gel visualization:

Gel Extraction and Purification

Equipment:

Consumables:

Gel excision, solubilization and DNA binding:

2. Wash:

3. DNA elution:

Ligation

Equipment:

Consumables:

PCR Clean-up

Equipment:

Consumables:

DNA binding:

Wash

DNA elution

Plasmid Isolation: Miniprep

Equipment:

Consumables:

Restriction Enzyme Digestion

Equipment:

Consumables:

SDS-PAGE

Equipment:

Consumables:

Resolving gel preparation

Stacking gel preparation

Sample preparation

Gel running

Staining

Destaining

Standard PCR

Equipment:

Consumables:

Media Preparation

1.LB (Lysogeny Broth)

Equipment:

Consumables:

2.SOB (Super Optimal Broth)

Equipment:

Consumables:

3.SOC

Equipment:

Consumables:

4. M9 Medium

Equipment:

Consumables:

Functional Test

can gene kill switch functional test

Equipment:

Consumables:

dap gene kill switch functional test

Equipment:

Consumables:

This page is about our protocols, you can find all the experimental process you need.
Enjoy!