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

Competent cell making:

1. Inoculate 500μl of overnight culture (Escherichia coli with pKD46) to 50ml of fresh SOB-Mg medium with 50μl 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 250μl (depends on the amount of the cell) of chilled 10% glycerol.

Electroporation:

1. Mix 50μl of the competent cell and 5μl 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 20μl 10% arabinose immediately after the pulse.

4. Incubate it for 1 hour at 37°C.

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

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

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 250μl (depends on the amount of the cell) of chilled 10% glycerol.

Electroporation:

1. Mix 50μl 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.

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)

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	2μl	10μl (2X)	12μl
Gel extraction	depends on how much DNA solution to be extracted	5μl (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 5μl 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.

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

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 = 100μl).

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 700μl 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 500μl 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 200μl 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 50μl 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.

Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. Centrifuge

Consumables:

1. T4 DNA ligase (NEB)

2. T4 DNA ligase buffer (NEB)

1. Set up the following reaction on ice:

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

2. Gently mix the reaction and centrifuge.

3. Incubate at 16°C overnight.

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

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

DNA binding:

1. Add MQ or ddH₂O to DNA solution until the volume reaches 100μl.

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

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

4. Place a spin column in a provided collection tube. Transfer 550μl 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 500μl 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 200μl 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 50μl 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.

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

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 250μl of suspension buffer and suspend bacterial cells by vortex.

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

5. Add 350μl 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 750μl of supernatant to spin column.

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

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

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)

1. On the ice, add all the components.

Double Digestion:

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

Single Digestion(Structure Check):

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

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.

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

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 12μl of pellet or supernatant (depends on necessity) and move to a new fresh eppendorf.

3. Add 3μl 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 3μl 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.

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 (10μM)

5. KOD FX polymerase

1. On the 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 2.5mM	0.8
Template	1
Forward primer 10μM	1
Reverse primer 10μM	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)	∞

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)

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

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: 100μl/100ml media

Chloramphenicol: 50μl/100ml media

Ampicillin: 100μl/100μl media