Chemical Transformation

Purpose: to transform DNA (plasmid) into competent cells

Material
LB broth, ice, selection plates, SOC, spreaders

Preperation steps

1. Obtain ice


2. Turn the heat block on


3. Put SOC in room temperature


4. Place selection plates into 37°C incubation

Methods

1. Thaw 50µL competent E. coli cells on ice for 10 minutes


2. Split the 50muL of bacteria into 3 tubes of 16uL or 4 tubes of 12uL and transfer to the labelled tubes placed on ice beforehand.


3. Add 2.5muL of Golden Gate Assembly product into split competent cells


4. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex


5. Place the mixture on ice for 30 minutes. Do not mix


6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix


7. Place on ice for 5 minutes. Do not mix


8. Pipette 950 µl of room temperature SOC into the mixture


9. Incubate at 37°C and 200-250 rpm for 60 minutes


10. Mix the cells thoroughly by flicking the tube and inverting


11. Spread the plates


• 50µL of transformation mixture onto one plate


• Remaining transformation mixture onto another plate


• Spread with spreader
12. Incubate overnight at 37°C with plates upside down or incubate at 27°C for 48 hours

Colony PCR

Purpose: To amplify the desired construct inside a colony ready for gel electrophoresis

Material
Dream Taq Green Master Mix, forward primer (10µM), reverse primer (10µM), selectin plates, water, thermocycler, PCR tubes/plates

Table 1. Material required for making the master mix for colony PCR

cPCR reaction contents	1X
DreamTaq Green Master Mix	10 µL
Forward primer (10 µM)	1 µL
Reverse primer (10µM)	1 µL
Resuspended colony	1 µL
water (nuclease free)	7 µL
Total volume	20 µL

Methods

1. Add a single colony of cells to 13 µL of PBS./li>
   
2. Put 1muL of the resuspended colony into the above recipe of PCR mixture


3. Spin down the PCR mixture with a centrifuge


4. Put the remaining 12uL PBS with resuspended colony in the fridge


5. Incubate in the thermocycler


• Initial denaturation: 95°C 1-3 minutes


• Cycles (35 cycles)
• Denaturation: 95°C 30 seconds
• Annealing: Tm 30 seconds (primer dependent, calculated with NEB Tm calculator)
• Extension: 72 °C (length dependent: 30s/kb)
• Final extension: 72°C 5-15 minutes
• Hold: 4°C Indefinitely
6. Gel electrophoresis


7. Transfer the 12uL of PBS with resuspended colony into LB liquid culture for overnight growth given colony PCR yields positive results after gel electrophoresis

Gel electrophoresis

Purpose: To investigate whether the DNA sample is of the correct length

Material

1X TAE, Agarose, SYBR Safe, Loading Buffer, Hyperladder, Casting tray, well combs, Voltage source, gel tank, suitable light source, microwave

Methods
Making agarose gel

1. For 1% gel, measure 1g of agarose per 100mL TAE and mix the appropriate amount of agarose and TAE/li>
   
2. Microwave and mix for 1-3 minutes until the agarose is completely dissolved


• Do not overboil


• Microwave in pulses and swirl occasionally
3. Cool for about 5 minutes


4. Add 1µL/10mL TAE of SYBR Safe and swirl until well mixed


5. Pour slowly the agarose into a gel tray with the well comb in place


6. Use a pipette tip to remove any bubbles or move any bubbles to the corner


7. Wait for about 30 minutes for the gel to set at the room temperature

Loading gel and electrophoresis

1. Add 1µL of loading buffer to 5 µL of DNA sample
• DreamTaq Green Master Mix contains loading dye thus this step is not required
2. Place the set gel into the gel tank with the comb side at the negative end of the gel tank and remove the comb carefully


3. Fill the gel tank with TAE to cover the comb holes


4. Add 8µL of 1kB hyperladder to the first well


5. Carefully load the remaining samples


6. Set the voltage to about 90V and press run


7. Wait until the dye is approximately 75-80% way down


8. Turn off and remove electrodes


9. Remove gel and visualise under blue light source

Electroporation

Purpose: To make incompetent cells electrically competent

Material

Electroporator (BioRad GenePulser), SOC, ice, e-cuvette, selection plates, spreaders

Methods
Glycerol washing

1. Inoculate a single colony of incompetent cells into 2 mL LB containing the appropriate antibiotics and incubate at 37°C overnight


2. Dilute overnight culture 1 mL into 25 mL LB containing appropriate antibiotics and shake at 37 °C until OD600 = 0.6-0.8 (1.5 hours to 3 hours)


3. Transfer the culture into 50 mL falcon tube


4. Spin down cells at 5000 rpm at 4°C for 10 minutes and resuspend pallet in 25 mL ice-cold 10% glycerol


5. Repeat step 4


6. Spin down cells at 5000 rpm at 4°C for 10 minutes


7. Resuspend pallet in 1 mL ice-cold 10% glycerol


8. Spin down cells at 14000 rpm at 4°C for 1 minute


9. Resuspend pallet in 250µL ice-cold 10% glycerol


10. Aliquot into ice-cold cyro tubes (Each 50µL) and store at -80°C

Electroporation

1. Thaw the prewashed glycerol stocks (50uL) on ice


2. Chill the e-cuvettes and slider on ice


3. Put 1µL of DNA into the stock tube containing glycerol stocks and wait for 5 minutes on ice


4. Transfer the entire volume inside the stock tube into a e-cuvette, make sure that the liquid is in contact with the metal wall


5. Wipe the e-cuvette and the slider to make sure no water droplet is on the wall


• If there is still water droplet, the pulse will cause spark which will damage the cells
6. Put the e-cuvette into the slider and slide the slider with the e-cuvette into the chamber of the electroporator


7. Start electroporation by pressing on the pulse button and hold for 5 seconds until a buzzing tone is heard. Release and slide the slider out of the chamber


8. Add 500µL of SOC into the e-cuvette immediately


9. Carefully transfer all the mixture from the e-cuvette into a 1.5mL tube


10. Incubate at 37°C and 200-250 rpm for 60 minutes


11. Spread the plates


• 50µL of transformation mixture onto one plate


• Remaining transformation mixture onto another plate


• Spread with spreader


12. Incubate overnight at 37°C with plates upside down or incubate at 27°C for 48 hours

JUMP Golden Gate Assembly

Purpose: To perform cloning to put DNA pieces together at a desired sequence

Level 0

Material

T4 ligase buffer, T4 ligase enzyme, BsmBI-v2/Esp3I, water, DNA inserts, DNA destination vector, PCR tubes

If using BsmBI-v2

Table 2. Material required for Level 0 JUMP Golden Gate Assembly with BsmBI-v2

JUMP Level 0 Contents	1X (10µL)
T4 ligase buffer	1 µL
T4 ligase enzyme	0.5 µL
BsmBI-v2	0.5 µL
water (Nuclease free)	6 µL
Desination vector (7.5nM)	1 µL
Insert (15nM)	1 µL
Total volume	10 µL

Thermocycler conditions
Non-overnight assembly protocol

• 42°C 15 minutes


• 30 cycles


• 42°C 3 minutes


• 16°C 3 minutes


• 55°C 15 minutes


• 80°C 5 minutes


• 4°C hold

Overnight assembly protocol

• 42°C 15 minutes


• 60 cycles


• 42°C 5 minutes


• 16°C 5 minutes


• 55°C 60 minutes


• 80°C 5 minutes


• 4°C hold

If using Esp3I

Table 3. Material required for Level 0 JUMP Golden Gate Assembly with Esp3I

JUMP Level 0 Contents	1X (10µL)
T4 ligase buffer	1 µL
T4 ligase enzyme	0.5 µL
Esp3I	1 µL
water (Nuclease free)	5.5 µL
Desination vector (7.5nM)	1 µL
Insert (15nM)	1 µL
Total volume	10 µL

Thermocycler conditions
Non-overnight assembly protocol

• 37°C 3 minutes


• 20 cycles


• 37°C 2 minutes 30 seconds


• 16°C 3 minutes


• 50°C 5 minutes


• 80°C 10 minutes


• 4°C hold

Overnight assembly protocol

• 37°C 3 minutes


• 40 cycles


• 37°C 9 minutes


• 16°C 12 minutes


• 55°C 5 minutes


• 80°C 10 minutes


• 4°C hold

Level 1

Material

T4 ligase buffer, T4 ligase enzyme, BsaI-HF, water, DNA inserts, DNA destination vector, PCR tubes

Table 4. Material required for Level 1 JUMP Golden Gate Assembly with BsaI-HF

JUMP Level 1 Contents	1X (10µL)
T4 ligase buffer	1 µL
T4 ligase enzyme	0.5 µL
BsaI-HF	0.5 µL
water (Nuclease free)	3 µL
Desination vector (7.5nM)	1 µL
Insert 1 (15nM)	1 µL
Insert 2 (15nM)	1 µL
Insert 3 (15nM)	1 µL
Insert 4 (15nM)	1 µL
Total volume	10 µL

Thermocycler conditions
Non-overnight assembly protocol

• 37°C 3 minutes


• 20 cycles


• 37°C 2 minutes 30 seconds


• 16°C 3 minutes


• 50°C 5 minutes


• 80°C 10 minutes


• 4°C hold

Overnight assembly protocol

• 37°C 3 minutes


• 40 cycles


• 37°C 9 minutes


• 16°C 12 minutes


• 55°C 5 minutes


• 80°C 10 minutes


• 4°C hold

Level 2

Material

T4 ligase buffer, T4 ligase enzyme, BsmBI-v2/Esp3I, water, DNA inserts, DNA destination vector, PCR tubes

If using BsmBI-v2

Table 5. Material required for Level 2 JUMP Golden Gate Assembly with BsmBI-v2

JUMP Level 2 Contents	1X (10µL)
T4 ligase buffer	1 µL
T4 ligase enzyme	0.5 µL
BsmBI-v2	0.5 µL
water (Nuclease free)	3 µL
Desination vector (7.5nM)	1 µL
Insert 1 (15nM)	1 µL
Insert 2 (15nM)	1 µL
Insert 3 (15nM)	1 µL
Insert 4 (15nM)	1 µL
Total volume	10 µL

Thermocycler conditions
Non-overnight assembly protocol

• 42°C 15 minutes


• 30 cycles


• 42°C 3 minutes


• 16°C 3 minutes


• 55°C 15 minutes


• 80°C 5 minutes


• 4°C hold

Overnight assembly protocol

• 42°C 15 minutes


• 60 cycles


• 42°C 5 minutes


• 16°C 5 minutes


• 55°C 60 minutes


• 80°C 5 minutes


• 4°C hold

If using Esp3I

Table 6. Material required for Level 2 JUMP Golden Gate Assembly with Esp3I

JUMP Level 2 Contents	1X (10µL)
T4 ligase buffer	1 µL
T4 ligase enzyme	0.5 µL
Esp3I	1 µL
water (Nuclease free)	2.5 µL
Desination vector (7.5nM)	1 µL
Insert 1 (15nM)	1 µL
Insert 2 (15nM)	1 µL
Insert 3 (15nM)	1 µL
Insert 4 (15nM)	1 µL
Total volume	10 µL

Thermocycler conditions
Non-overnight assembly protocol

• 37°C 3 minutes


• 20 cycles


• 37°C 2 minutes 30 seconds


• 16°C 3 minutes


• 50°C 5 minutes


• 80°C 10 minutes


• 4°C hold

Overnight assembly protocol

• 37°C 3 minutes


• 40 cycles


• 37°C 9 minutes


• 16°C 12 minutes


• 55°C 5 minutes


• 80°C 10 minutes


• 4°C hold

Downstream site (DSS) integration

Material

T4 ligase buffer, T4 ligase enzyme, BbsI, water, linear ligated DNA, DNA destination vector, PCR tubes

Table 4. Material required for DSS JUMP Golden Gate Assembly with BbsI

JUMP DSS integration Contents	1X (10µL)
T4 ligase buffer	1 µL
T4 ligase enzyme	0.5 µL
BbsI	1 µL
water (Nuclease free)	5.5 µL
Desination vector (7.5nM)	1 µL
linear ligated DNA (15nM)	1 µL

Thermocycler conditions
Non-overnight assembly protocol

• 37°C 3 minutes


• 20 cycles


• 37°C 2 minutes 30 seconds


• 16°C 3 minutes


• 50°C 5 minutes


• 80°C 10 minutes


• 4°C hold

Overnight assembly protocol

• 37°C 3 minutes


• 40 cycles


• 37°C 9 minutes


• 16°C 12 minutes


• 55°C 5 minutes


• 80°C 10 minutes


• 4°C hold

Linear Golden Gate Assembly

Purpose: to linear ligate fragments of DNA together to form transcription units and adapters on either sides for subsequent assemblies

Linear ligation
Material
DNA adapter, DNA fragments, DNA ligase buffer, DNA ligase, BsaI-HF, nuclease free water, PCR tubes, thermocycler

Table 7. Material required for linear Golden Gate Assembly with BsaI-HF

linear Golden Gate Assembly Contents	1X (10µL)
T4 ligase buffer	1 µL
T4 ligase enzyme	0.5 µL
BsaI-HF	0.5 µL
water (Nuclease free)	2 µL
5' adapter (25nM)	1 µL
Promoter (15nM)	1 µL
RBS (15nM)	1 µL
CDS (15nM)	1 µL
Terminator (15nM)	1 µL
3' adapter (25nM)	1 µL
Total volume	10 µL

Methods
Thermocycler Conditions

• Cycles (9 cycles)


• 37°C 3 minutes


• 16°C 4 minutes


• 50°C 5 minutes


• 4°C hold

Post linear ligation amplication
Materials
Q5 PCR Hot Start Master Mix, forward primer, reverse primer, nuclease free water, Golden Gate Assembly product, PCR tube, thermocycler, materials for gel electrophoresis

Table 8. Material required for Q5 PCR after linear Golden Gate Assembly

Q5 PCR amplication contents	1X (50µL)
2X Q5 Hot Start Master Mix	25 µL
Forward primer (10µM)	2.5 µL
Reverse primer (10µM)	2.5 µL
water (Nuclease free)	18.5 µL
Golden Gate Assembly Product	1.5 µL
Total volume	50 µL

Methods

1. Add the above recipe into PCR tubes


2. Spin down with a centrifuge


3. Thermocycler Conditions


• 98°C 30 seconds


• Cycles (30 cycles)


• 98°C 15 seconds


• Tm 30 seconds (calculated with NEB Tm calculator)


• 72°C 20 seconds/kB


• 72°C 5 minutes
• 4°C hold
4. After PCR, transfer 5μL for gel electrophoresis to check the band size (Refer to the gel electrophoresis protocol)

DNA purification


Refer to the PCR product purification protocol

Making glycerol stocks

Purpose: Create stocks of bacteria with desired plasmid for long-term storage

Material

Cyro tubes, 50% glycerol, bacterial culture

Methods

1. 500μL of culture is added to 500μL of 50% glycerol in cyro tube


• Note: make 2 glycerol stocks per sample
2. Pipette up and down the mixture to mix


3. Put the glycerol stocks into the -80°C freezer

Miniprep

Purpose: To extract plasmid DNA from bacteria

Material

ThermoFischer MiniPrep Kit, 1.5mL tubes

Methods
All centrifuge steps are carried out at 17000g

1. Transfer all the remaining culture from snapcaps into 15mL falcon tubes


2. Centrifuge at 5000rpm for 5 minutes


3. Discard all supernatent
4. Add 250μL of Resuspension buffer into the tube and vortex until well mixed


5. Transfer all the solution into a 1.5mL tube
6. Add 250μL of Lysis solution and invert 4-6 times (leave for 1-3 minutes)


• Do not wait for more than 5 minutes
7. Add 350μL of Neutralisation solution and invert 4-6 times and wait for 5 minutes


8. Centrifuge for 10 minutes
9. Transfer the supernatent into the spin column, ensure not to take any pallet. If the pallet becomes dislodged, repeat the centrifuge step


10. Centrifuge for 1 minute
11. Wash with 500μL wash solution and wait for 5 minutes, centrifuge for 1 minutes and discard flow through


12. Repeat step 11
13. Transfer the column into a new empty 1.5 mL tube then centrifuge for 10 minutes to remove remaining wash solution


14. Transfer the spin column into a new 1.5 mL tube
15. Add 36μL of warmed elution buffer and incubate for 2 minutes
16. Centrifuge for 2 minutes to elute the samples


17. Measure the yield and purity on Nanodrop

Oligo Annealing

Purpose: to anneal 2 single strand complementary DNA together into double stranded

Material

Primers for oligo annealing (100μM), water, Thermocycler, PCR tubes

Methods

1. Take 1μL of each primer and add it into 8μL of water into PCR tubes


2. Spin down with a centrifuge


3. Thermocycler protocol


• 95°C 2 minutes


• -1°C/min until 10°C


• Hold at 4°C indefinitely


4. PCR product purification

Q5 PCR

Purpose: to amplify template DNA with minimal error rate

Materials
Q5 PCR Hot Start Master Mix, forward primer, reverse primer, nuclease free water, template DNA, PCR tube, thermocycler

Table 9. Material required for 25µL Q5 PCR reaction

Q5 PCR amplication contents	1X (25µL)
2X Q5 Hot Start Master Mix	12.5 µL
Forward primer (10µM)	1.25 µL
Reverse primer (10µM)	1.25 µL
water (Nuclease free)	9 µL
Template DNA	1 µL
Total volume	25 µL

Methods

1. Add the above recipe into PCR tubes


2. Spin down with a centrifuge


3. Thermocycler Conditions


• 98°C 60 seconds


• Cycles (35 cycles)


• 98°C 15 seconds


• Tm 15 seconds (calculated with NEB Tm calculator)


• 72°C 20 seconds/kB


• 72°C 2 minutes
• 4°C hold

PCR purification

Purpose: To purify and concentrate DNA Product after PCR

Material

DNA Clean & Concentrator (Zymogen), 1.5mL tubes

Methods

All centrifugation steps should be performed 16,000 x g.

1. Add the 5 volumes of DNA Binding Buffer to each volume of DNA sample. Mix briefly by vortexing.


2. Transfer the mixture to a spin column with a collection tube


3. Centrifuge for 30 seconds, discard flow-through


4. Add 200 µl DNA Wash Buffer into the column and centrifuge for 30 seconds, discard flow through


5. Repeat step 4


6. Add 10 µl Elution Buffer into the column and incubate for 2 minutes


7. Transfer the column into a 1.5mL tube and centrifuge for 30 seconds to elute the DNA
8. Use nanodrop to measure the concentration of DNA product

Making LB agar selection plates

Purpose: To make selection plates for plating of bacteria

Material

500mL bottle, LB agar, water, autoclave, petri dishes, Biological Safety Cabinet

Methods

1. Add 16g of LB Agar Miller into 400 mL of deionised water into the 500 mL bottle


• never fill the whole 500 mL bottle completely
2. Shake the bottle until well-mixed


3. Autoclave the bottle


• Loosen the bottle lid
• Stick Autoclave tape onto the bottle
4. After autoclaving, take the bottle out out of the autoclave and allow it to cool
5. In the Biological Safety Cabinet, add in 1 in 1000 dilution of 1000X appropriate antibiotics if necessary
6. Pour about 20-25 mL of LB agar into the petri dishes
7. Wait for the agar to solidy and store it in the 4°C fridge

Making LB broth medium

Purpose: To culture bacteria overnight

Material

500mL bottle, LB broth, water, autoclave

Methods

1. Add 10g of LB Broth Miller into 400 mL of deionised water into the 500 mL bottle


• never fill the whole 500 mL bottle completely
2. Shake the bottle until well-mixed


3. Autoclave the bottle


• Loosen the bottle lid
• Stick Autoclave tape onto the bottle
4. After autoclaving, take the bottle out out of the autoclave and allow it to cool
5. Store the bottle in room temperature