Materials:

• 2×Phanta Flash Master Mix or 2×Santaq Fast PCR Mix

• DNA Template

• Forward primer and reverse primer (10 μM)

Procedures:

1. Assemble all reaction components in PCR tubes. Assembly is done on ice. The components used for PCR are shown in Table 1.

   Component	50 μl reaction	100 μl reaction	Final concentration
   2×Phanta Flash Master Mix or 2×Santaq Fast PCR Mix	25 μL	50 μL	1×
   DNA Template	variable	variable	~20 ng
   Forward Primer (10μM)	2 μL	2 μL	0.4 μM
   Reverse Primer (10μM)	2 μL	2 μL	0.4 μM
   Nuclease-Free Water	to 50 μL	to 100 μL	-

   [^Table 1]: Components of PCR mixture.

2. Collect all the liquid to the bottom of the tubes by centrifuging for a few seconds.

3. Place the PCR tubes into a thermocycler and run the program in Table 2.

   Step	Temperature	Time
   1 Cycle	Initial Denaturation	98 $^\circ C$	30 s
   30 Cycle	Denaturation	98 $^\circ C$	10 s
   	Annealing	Refolding Temperature (Tm－5 $^\circ C$ & lower than 60 $^\circ C$)	5 s
   	Extension	72 $^\circ C$	4-5sec/kb
   1 Cycle	Final Extension	72 $^\circ C$	7 min
   1 Cycle	Hold	4 $^\circ C$	$\infty$

   [^Table 2]: PCR Program.

Colony PCR ​

Materials:

• 2×Santaq Fast buffer

• Colonies from liquid cultures

• Forward primer and reverse primer (10 μM)

Procedures:

1. Assemble all reaction components in PCR tubes. The components used for PCR are shown in Table 3.

   Component	10 μl reaction	20 μl reaction
   2×Santaq Fast buffer	5 μL	10 μL
   Colonies from liquid cultures	1 μL	2 μL
   10μM Forward primer	0.5 μL	1 μL
   10μM Reverse primer	0.5 μL	1 μL
   Nuclease-Free Water	3 μL	6 μL

   [^Table 3]: Components of cPCR reaction system.

2. Collect all the liquid to the bottom of the tubes by centrifuging for a few seconds.

3. Place the PCR tubes into a thermocycler and run the program in Table 4.

   Step	Temperature	Time
   1 Cycle	Initial Denaturation	98 $^\circ C$	30 s
   30 Cycle	Denaturation	98 $^\circ C$	10 s
   	Annealing	55 $^\circ C$	5 s
   	Extension	72 $^\circ C$	4-5sec/kb
   1 Cycle	Final Extension	72 $^\circ C$	7 min
   1 Cycle	Hold	4 $^\circ C$	$\infty$

   [^Table 4]: Colony PCR Program.

Materials:

• Two single-stranded oligonucleotides with complementary sequences (100 μM)
• T4 PNK
• Ligation buffer
• Ligase

Procedure:

1. Add all reaction components into PCR tubes. The components are demonstrated in Table 5.

   Component	10 μl reaction
   Ligation Buffer	2 μL
   Ligase	0.5 μL
   Oligo 1 (100μM)	1 μL
   Oligo 2 (100μM)	1 μL
   T4 PNK	0.5 μL
   Nuclease-Free Water	5 μL

   [^Table 5]: Components of oligo annealing reaction system.

2. Collect all the liquid to the bottom of the tubes by centrifuging for a few seconds.

3. Place the PCR tubes into a thermocycler and run the program in Table 6.

   Temperature	Time
   37 $^\circ C$	30 s
   95 $^\circ C$	1 min
   4 $^\circ C$ (slowly cool at the speed of 0.8$^\circ C$/s )	$\infty$

   [^Table 6]: Oligo annealing program.

4. Dilute the concentration of DNA to 50-100ng/ul.

Through Restriction Enzyme Digestion ​

Materials:

• The vector which is to be linearized

• Restriction enzymes

• Reaction buffer supplied by NEB

Procedures:

1. Add all reaction components into PCR tubes. The components are demonstrated in Table 7.

   Component	50 μl reaction
   DNA Vector	2-3 μg
   Restriction enzymes	1 μL for each (no more than one tenth of the whole system)
   Reaction Buffer*	5 μL
   Nuclease-Free Water	to 50 μL

   [^Table 7]: Linearization reaction mixture. *Reaction Buffer is based on https://nebcloner.neb.com/#!/redigest.

2. Collect all the liquid to the bottom of the tubes by centrifuging for a few seconds.

3. Place the PCR tubes into a thermocycler and run the program in Table 8.

   Temperature	Time
   37 $^\circ C$	1 - 4 h
   Deactivation Temperature*	20 min
   4 $^\circ C$	$\infty$

   [^Table 8]: Linearization Program. *Deactivation Temperature is based on https://nebcloner.neb.com/#!/redigest.

Through PCR ​

The vecter is used as a DNA template, and the other steps are the same as normal PCR.

Materials:

• Agarose

• TAE buffer

• 4S green plus dye

• 10×DNA loading buffer Procedures:

Pouring a Gel ​

1. Mix 1×TAE buffer and agarose according to the concentration of the required gel (typically 1%).
2. Microwave for 1-3 min until the agarose is completely dissolved.
3. Cool the solution at room temperature until it is slightly hot and add 4S green plus to a concentration of 0.1 μL/mL.
4. Stir with a magnetic stirrer
5. Pour solution into mold and wait for 30-60 minutes
6. Remove the gel, cover it with plastic wrap
7. Store it at 4 $^\circ C$

Running a Gel ​

1. Place the agarose gel into the gel box (electrophoresis unit).
2. Fill gel box with 1xTAE (or TBE) until the gel is covered.
3. Add loading buffer to each of DNA samples.
4. Load a molecular weight ladder into the first lane of the gel.
5. Load samples into other wells of the gel.
6. Run the gel at 80-160 V. Run time is about 0.5-1.0 hours, depending on the gel concentration and voltage.

Visualising a Gel ​

1. View the gel under a UV or blue light transilluminator.

Materials:

• Distilled water

• DNA Sample

• Blanking Sample

Procedures:

1. Clean both pedestals with a new laboratory wipe, pipette 2 µL of distilled water onto the lower pedestal, lowering the arm, waiting a few seconds, then clean both pedestals again.
2. Pipette 2 µL blanking solution onto the pedestal and lower the arm.
3. Lift the arm and clean both pedestals with a new laboratory wipe, then pipette 1-2 µL sample solution onto the pedestal and lower the arm to start the sample measurement.
4. If there are more than one sample, back to step three.

Materials：

• 2×CE Mix

• Lineared DNA vector

• DNA fragments

Procedures:

1. The concentration determination of linearized vectors and inserts with Nanodorp.

2. The calculation of vectors and inserts usage

   For single-fragment homologous recombination, the optimal amount of vector required is 0.03 pmol, the optimal amount of insert required is 0.06 pmol (the molar radio of vector to insert is 1:2). For multi-fragment homologous recombination, the optimal amount of inserts and linearized vectors are both 0.03 pmol (the molar radio of vector to insert is 1:1). These mass can be roughly calculated according to the following formula:

   Single-fragment homologous recombination The optimal mass of vector required = [0.02 × number of base pairs] ng (0.03 pmol) The optimal mass of insert required = [0.04 × number of base pairs] ng (0.06 pmol)

   Multi-fragment homologous recombination The optimal mass of vector required = [0.02 × number of base pairs] ng (0.03 pmol) The optimal mass of each insert required = [0.02 × number of base pairs] ng (0.03 pmol)

   For single-fragment homologous recombination: The mass of amplified insert should be more than 20 ng. When the length of the insert is larger than that of the vector, the calculation method of the optimal mass of vector and insert should be inverted. For multi-fragment homologous recombination: The mass of each insert should be more than 10 ng. When the optimal mass calculated by the above formula is below 10 ng, just use 10 ng directly.

3. Add all reaction components into PCR tubes. Assembly is done on ice. The components are demonstrated in Table 9.

   Components	10 μl reaction
   2×CE Mix	5 μL
   Lineared vector	variable
   Fragments	variable
   Nuclease-Free Water	to 10 μL

   [^Table 9]: Components of gibson assembly system.

4. Collect all the liquid to the bottom of the tubes by centrifuging for a few seconds.

5. Incubate it in 50 $^\circ C$ for 1 h and immediately chill the tube at 4°C or on ice.

6. Add the formulated system into competent cell and then finish transformation experiment.

Materials:

• Competent cells

• vector

• Liquid LB media

• Solid LB agar plates

Procedures:

1. Thaw competent cells on ice.
2. Chill approximately 5 ng (typically 5-10 μL) of the vector in a 1.5 mL micro-centrifuge tube.
3. Add 100 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes.
5. Heat shock at 42°C for 30 seconds. Do not mix.
6. Replace on ice for 2-3 minutes
7. Add 900 µl of room temperature LB media to the tube.
8. Place tube at 37°C for 60 minutes. Shake vigorously (220 rpm).
9. Warm selection plates to 37°C.
10. Spread 25–50 µl of the cells and ligation mixture onto the plates.
11. Incubate overnight at 37°C.

Materials:

• Tryptone

• Yeast Extract

• Sodium Chloride (NaCl)

• Agar (optional for solid medium)

Procedures:

1. Accurately weigh out required amount of tryptone, yeast extract and NaCl.

2. Add the weighed ingredients to an required amount of water. Take 1000 mL LB media as example, the formulation is shown in Table 10 below.

   Components	Amount
   Tryptone	10 g
   Yeast Extract	5 g
   NaCl	10 g
   Agar (optional for solid medium)	10 - 15 g
   H₂O	1000 mL

   [^Table 10]: Formulation of LB medium.

3. Place the medium solution in an autoclave and sterilize at 120 $^\circ C$ for 20 minutes. (We turned to our lab professionals for help with sterilization and avoided doing it ourselves.)

4. (optional) After sterilization, the medium will be stored in 4 $^\circ C$. For the selection of bacteria cells after transformation, antibiotics were added to the media in the following concentrations: 32 ug/mL of chloramphenicol, 50 ug/mL of kanamycin or 100 ug/mL of ampicillin.

Procedures:

1. The bacteria were grown in 5 mL of LB media in shaker (220 rpm, 37 $^\circ C$) until they reached an OD600 above 0.75 (typically longer than 20 h).
2. 100 μL - 150 μL of the cell suspension were transferred into their designated wells of the 96-well plate.
3. For the analysis, a BioTek Synergy Mx Microplate Reader equipped with a 587 nm wavelength LED for mCherry excitation was utilized.

Materials:

• FastPure Plasmid Mini Kit

• Bacterial broth

Procedure：

1. Take 1-5 mL of overnight culture (12-16 hours) of bacterial broth, centrifuge at 10,000 rpm (equivalent to 11,500 × g) for 1 minute, and discard the culture medium.
2. Add 250 μL of Buffer P1, and mix well with a pipette.
3. Add 250 μL of Buffer P2, and gently invert to mix 8-10 times.
4. Add 350 μL of Buffer P3, and immediately mix gently by inverting 8-10 times. After a white flocculent precipitate appears, centrifuge at 12,000 rpm (13,400 × g) for 10 minutes.
5. Transfer the supernatant to an adsorption column, and centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds, then discard the waste liquid.
6. Add 600 μL of Buffer PW2 to the adsorption column. Centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds, and discard the waste liquid. (Repeat this step twice.)
7. Centrifuge at 12,000 rpm (13,400 × g) for 1 minute to dry the adsorption column.
8. Place the adsorption column into a new 1.5 mL centrifuge tube, add 50 μL of nuclease-free water. Let it stand at room temperature for 2 minutes, then centrifuge at 12,000 rpm (13,400 × g) for 1 minute to elute the DNA.
9. Discard the adsorption column, and store the DNA product at -20°C.

Materials:

• Buffer GDP: DNA binding buffer.

• Buffer GW: Washing buffer; add absolute ethanol by the indicated volume on the bottle before use.

• Elution buffer: Elution, can be replaced by Nuclease-Free Water.

• The gel containing the target DNA fragment or DNA fragments from PCR.

Procedures:

Gel Extraction Program ​

1. After DNA electrophoresis is complete, quickly excise the gel containing the target DNA fragment under UV light, blot away any liquid from the surface with a paper towel, and mince the gel. Weigh the gel, with 100 mg of gel being equivalent to a volume of 100 μl.

2. Add an equal volume of Buffer GDP. Incubate at 50-55°C in a water bath for 7-10 minutes to ensure the gel piece is completely dissolved. During the incubation, invert the tube twice to facilitate dissolution.

3. Briefly centrifuge to collect any droplets on the walls of the collection tube, and transfer up to 700 μl of the dissolved gel solution to the adsorption column, centrifuging at 12,000 rpm (13,800 × g) for 30-60 seconds.

4. Discard the flow-through. Add 300 μl of Buffer GDP. Let it stand for 1 minute. Centrifuge at 12,000 rpm (13,800 × g) for 30-60 seconds.

5. Discard the flow-through. Add 700 μl of Buffer GW. Centrifuge at 12,000 rpm (13,800 × g) for 30-60 seconds. (Repeat this step twice.)

6. Discard the flow-through. Centrifuge at 12,000 rpm (13,800 × g) for 2 minutes.

7. Place the adsorption column into a 1.5 ml centrifuge tube, and add 30 μl of elution buffer or nuclease-free water to the center of the adsorption column. Let it stand for 2 minutes. Centrifuge at 12,000 rpm (13,800 × g) for 1 minute. Store the DNA at -20°C.

PCR Products Recovery Program ​

1. Briefly centrifuge PCR products, enzymatic reaction solution, and other DNA crude products. Estimate their volume with pipette and transfer to a sterilized 1.5 ml or 2 ml tube. Add nuclease-free water until the volume up to 100 μl; while for genomic DNA with high concentration, diluting to 300 μl with nuclease-free water will help to improve recovery efficiency.

2. Add 5 volumes of Buffer GDP, mix thoroughly by inverting or vortexing. If DNA fragment of interest ≤100 bp, additional 1.5 volumes (samples + Buffer GDP) of ethanol need to be added.

3. Insert the column back into the collection tube, transfer the mixtrue to the column, centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 sec. If the volume of the mixed solution is >700 µl, put the adsorption column back into the collection tube, transfer the remaining solution to the adsorption column, and centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 sec.

4. The next performance refers to the step 5 - 7 of Gel extraction program.

Follow the instruction of the kits M1100S offered by NEB. (https://www.neb.com/en/protocols/2021/09/14/protocol-for-nebridge-ligase-master-mix-neb-m1100)

Procedures:

1. Determine reaction component volumes, as shown in Table 11 and Table 12. Set up a reaction in a microcentrifuge tube on ice and add all components.

   Components	Amount
   NEBridge Ligase Master Mix	5 µL
   DNA Fragments	0.05 pmol each
   Type IIS Restriction Enzyme (Follow Table 2 in kit protocol)	X μL
   nuclease-free water	to 15 μL

   [^Table 11]: Reaction system of Golden Gate Assembly.

2. Incubate for the recommended time and temperature. (Follow Table 3 in kit protocol)

3. End Soak: Incubate at 60°C for 5 minutes, before transformation.

4. Chill on ice.

5. Use 2 μl of the reaction to transform 50 μl of competent cells. If reaction will not be used immediately for transformation, store at -20°C.