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. 10 M NaOH
6. Agar (for LB agar)
7. Antibiotics (for selection plate)

Protocol

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

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

2. Mix well, and pour the mixture into the bottle or flask and autoclave. Notes: For agar medium, pour the mixture into the petri dish and dry the plates.

For selection plates, add antibiotics as the following before pouring the mixture into the petri dish:

1. Kanamycin: 100 μL/100 mL media
2. Chloramphenicol: 50 μL/100 mL media
3. Ampicillin: 100 μL/100 mL media

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. 5 M NaCl
5. 3 M KCl

Protocol

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

   Components	Volume
   DW	96 mL
   Tryptone	2 g
   Yeast Extract	0.5 g
   5 M NaCl	0.2 mL
   3 M KCl	83 μL
   Total Volume	100 mL

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

Materials:

Equipment:

    Pipette and pipette tips

Consumables:

1. SOB medium in a glass bottle
2. 1 M MgCl₂
3. 1 M MgSO₄
4. 20% glucose

Protocol

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

Materials

Equipment:

    Pipette and pipette tips

Consumables:

1. M9 salts (5X)
2. 20% glucose
3. 1 M MgSO₄
4. 1 M CaCl₂
5. H₂O

Protocol

1. Add chemicals as the following to a bottle.

   Components	Volume
   M9 salts (5X)	20 mL
   20% glucose	2 mL
   1 M MgSO4	200 μL
   1 M CaCl2	10 μL
   H2O	78 mL
   Total Volume	100 mL

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

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Centrifuge
4. Hot plate
5. OD meter

Consumables:

1. 0.4 M Boric acid buffer (pH 10)
2. 6% Phenol
3. 6% Sodium hypochlorite (NaClO)
4. Overnight cultured bacteria (with plasmids encoded gadB gene)

Protocol

1. Add 1.5 mL overnight cultured bacteria to a 1.5 mL microfuge tube.
2. Centrifuge at 14000 rpm for 3 minutes.
3. In a 2 mL eppendorf add all the components below in order, making sure that phenol is being added in the fume hood.

   Components	Volume
   0.4 M Boric acid buffer (pH 10)	100 μL
   Supernatant of bacteria	10 μL
   6% Phenol	100 μL
   6% Sodium hypochlorite (NaClO)	100 μL

4. After adding all the components, place the microfuge at room temperature for 5 minutes.
5. Put it onto the hot plate at 100℃ for 10 minutes.
6. Load 200 μL of the sample and the standard into 96 wells.
7. Test the OD value at 630 nm by OD meter and draw the standard curve to later calculate the concentration of GABA.

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Culturing plate
4. OD meter

Consumables:

1. LB medium
2. Overnight cultured bacteria (with plasmids encoded gadB gene and melA gene)

Protocol

The experiment was divided into three parts: UV light exposure, dilution and dropping.

UV light exposure

1. Expose the plates which have bacteria colonies to UV light for 5 minutes.
2. Wash down the bacteria colonies with LBYT medium.
3. Adjust the OD values of LBYT medium containing bacteria to 1.

Dilution

Dilutes the samples 1~10^-6 times

Dropping

Drop 5 μL of each sample onto the agar plates, and culture them overnight.

Materials

Equipment:

1. PCR tubes
2. Ice bucket
3. Thermocycler
4. Pipette and pipette tips

Consumables:

1. Sterilized ddH₂O
2. 10X PCR buffer for Ex Taq
3. 2 mM dNTP
4. Forward and reverse primer (10 μM)
5. Ex Taq polymerase

Protocol

1. Add the following chemicals to the PCR tube on ice:

   Components	Volume (μL)
   Template DNA	1
   10X PCR buffer for Ex Taq	5
   2 mM dNTP	5
   Primer (forward and reverse)	2
   Ex Taq polymerase	0.1
   Sterilized ddH2O	36.9
   Total Volume	50

2. Gently mix the PCR solution and centrifuge briefly.
3. Transfer the PCR tubes to a thermocycler.

   Step	Temperature (℃)	Time (second)
   Initial denaturalization	94	300
   25 - 35 cycles	94 (denaturation)	30
   	55 (annealing)	30
   	72 (extension)	60
   Final extension	72	600
   Hold	16	infinity

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Ice bucket
4. Incubator (37℃)

Consumables:

1. Restriction enzymes
2. 10X Buffer (depends on its restriction enzymes)
3. DNA sample
4. MQ or ddH₂O (for single digestion)

Protocol

1. On the ice, add all the following components.
   Double Digestion:

   Components	Volume
   DNA	43 μL
   10X Buffer	5 μL
   Restriction enzyme 1	1 μL
   Restriction enzyme 2	1 μL
   Total Volume	50 μL

   Single Digestion (Structure Check):

   Components	Volume
   Plasmid DNA	2 μL
   10X Buffer	1.5 μL
   Restriction enzyme	0.5 μL
   MQ or ddH2O	11 μL
   Total Volume	15 μL

2. Mix gently and incubate for 3 hours for double digestion or 1 hour for single digestion at 37℃.
   Note: Incubation time varies along the total volume of the reaction.

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Ice bucket

Consumables:

1. T4 DNA ligase (Takara Bio)
2. 10X T4 DNA ligase buffer (Takara Bio)

Protocol

1. Add the following chemicals to the PCR tube on ice:

   Components	Volume
   Vector	2 μL
   Insert	6 μL
   10X buffer	1 μL
   T4 ligase	1 μL
   Total Volume	10 μL

2. Gently mix the reaction and centrifuge.
3. Incubate at 4℃ overnight.
4. Keep the DNA solution in the -20℃ freezer.

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Ice bucket
4. Centrifuge

Consumables:

1. Competent cell
2. IPTG/X-Gal
3. Plasmid DNA or Ligation product
4. Lysogeny broth (LB)
5. LB agar with Ampicillin (Amp)

Protocol

1. Thaw frozen competent cells on ice.
2. Add 10 μL ligation product or 1 μL plasmid DNA per 50 μL tube of competent cells, and quickly flick the tube several times.
3. Keep tubes on ice for 30 minutes.
4. Heat-shock the cells for 45 seconds in a water bath at exactly 42℃.
5. Immediately place the tubes on ice for 1 minute.
6. Add 1 mL LB medium to each transformation reaction and incubate at 37℃ for 2 hours at 180 rpm in a shaking incubator.
7. Centrifuge for 4 minutes at 4℃ and 5000 xg.
8. Discard the supernatant (about 800 μL to 1 mL) and keep a 200 μL medium.
9. Add 50 μL IPTG/X-Gal (IPTG:X-Gal; 1:4 premix), and resuspension.
10. For each transformation reaction, take 100 μL transformation product to LB agar plate with Amp and spread evenly.
11. Incubate the plates at 37℃ for 12-14 hours or overnight.

Materials

Equipment:

1. PCR tubes
2. Pipette and pipette tips
3. Ice bucket
4. Centrifuge

Consumables:

1. Nuclease free water
2. Ribomax 2X buffer
3. Enzyme mix
4. DNase
5. Isopropanol
6. Phenol (pH 4.3)
7. NaOAC
8. Chloroform + isoamyl alcohol (24:1 premix)
9. 75% EtOH
10. DEPC-treated water

Protocol

1. On the ice, add all components in a PCR tube, making up to 20 μL volume reaction.

   Components	Volume
   DNA template (1 μg/μL)	1 μL
   Nuclease-free water	7 μL
   Enzyme mix	2 μL
   Ribomax 2X buffer	10 μL
   Total Volume	20 μL

2. Gently mix the PCR reactions and centrifuge briefly.
3. Synthesis reaction for 3 hours at 37℃.
4. Reanneal tRNA for 20 minutes at 70℃.
5. Cold down for 30 minutes at room temperature.
6. Add 2 μL DNase to degrade DNA template for 15 minutes at 37℃.
7. Add phenol 52.5 μL and chloroform + isoamyl alcohol 10.5 μL in each tube
8. Vortex 1 minute, and Centrifuge for 2 minutes at 4℃ and 13,000 rpm.
9. Take the supernatant to the new tube, add chloroform + isoamyl alcohol 42 μL.
10. Vortex 1 minute, and Centrifuge for 2 minutes at 4℃ and 13,000 rpm.
11. Take the supernatant to the new tube, add 4.2 μL 3 M Sodium acetate, 42 μL isopropanol, on ice for 5 minutes.
12. Centrifuge for 10 minutes at 4℃ and 15,000 rpm.
13. Discard the supernatant, add 1 mL 75% EtOH to wash, discard EtOH.
14. Add 1 mL 75% EtOH, Centrifuge for 10 minutes at 4℃ and 13,000 rpm.
15. Discard the supernatant, in the biological safety cabinet (BSC) dry out RNA.
16. Add 104 μL DEPC-treated water to reconstitution RNA.

Before aminoacylation, sample pretreatment to refold tRNA correctly

tRNA reannealing

1. Dilute the sample two times.
2. Heat the solution at 95℃ for 2 min.
3. 20~22℃ for 3 min.
4. 37℃ for 5 min.
5. Put the samples on ice and cool down.
6. Collect the samples and store at -80℃.

Note: To prevent the sample from degrading, do not heat or cool the sample gradually.

Materials

Equipment:

1. PCR tubes
2. Pipette and pipette tips
3. Ice bucket
4. Centrifuge

Consumables:

1. Purified tRNA
2. Amino acid
3. 1 M HEPES-KOH (pH 7.6)
4. 100 mM ATP (pH 7)
5. 100 mM DTT
6. 3 M KCl
7. 1 M MgCl₂
8. Aminoacyl-tRNA synthetase
9. 3 M NaOAc (pH 5.2)
10. Phenol saturated with 300 mM NaOAc (pH 5.2)
11. CHCl₃ and isoamyl alcohol (24:1 premix)
12. 75% ethanol
13. 100% ethanol
14. YM-10 column

Protocol

1. Mix: In a reaction volume of 500 μL, combine the following components: 120 ug tRNA, buffer [100 mM HEPES-KOH (pH 7.6), 20 mM MgCl₂, 10 mM KCl, 1 mM DTT], 10 mM ATP, 100 uM amino acid, 1 mM AARS.
2. Incubate: Incubate the reaction in a circulating water bath at 37℃ for 20 minutes.
3. Extraction:
   1. Add 50 μL of 3 M NaOAc (pH 5.2).
   2. Extract with an equal volume of phenol for 10 minutes using a vortex-mixer in the cold room.
   3. Separate the phases by spinning them in a microfuge for 5 minutes at 13000 rpm. Move the aqueous phase to a new microfuge tube.
   4. Back extract the phenol phase with 300 mM NaOAc (pH 5.2), spin, remove the aqueous phase and combine it with the aqueous fraction saved from step (3).
   5. Repeat step (3), (4).
   6. Extract the aqueous phase twice with a mixture of CHCl₃ and isoamyl alcohol (24:1 premix).
4. Precipitate:
   1. Add 2.5 volumes of 100% ethanol to precipitate the aminoacyl-tRNA.
   2. Centrifugal for 15 minutes at 4℃ and 13000 rpm, and remove supernatant.
5. Wash pellet: wash with 75% ethanol
6. Reconstitution: Dissolve pellets in 20 μL of 2 mM NaOAc (pH 5.2).
7. Pass the aminoacyl-tRNA through spin columns and centrifuge for 30 min at 4℃ and 10000 xg.
8. Collect the solution above the membrane and store at -80℃.

Materials

Equipment:

1. Incubator
2. Vacuum dryer
3. Thermocycler
4. PCR tubes
5. Pipette and pipette tips
6. Ice bucket
7. Centrifuge

Consumables:

1. 100 mM Hepes, pH 7.0 (2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid)
2. 10 mM MgCl₂
3. 20 mM KCl
4. 1 mM dithiothreitol
5. 3 M potassium acetate (pH 4.6)
6. 75% ethanol
7. 100% ethanol
8. ddH₂O
9. ATP
10. Sodium selenite
11. SelD
12. SelA

Protocol

1. Prepare buffer H: 100 mM Hepes (pH 7.0), 10 mM MgCI₂, 20 mM KCl, 1 mM dithiothreitol
2. 70 pmol of seryl-tRNA were incubated for 30 minutes at 25℃ in 50 μL of buffer H with 22 pmol of purified enzyme (SelA), 70 pmol of purified SelD protein, 0.1 μmol of ATP, and 1 nmol of sodium selenite. (When adding the sodium selenite, it should be carried out in the hood.)
3. At the time intervals given, the reaction is stopped by the addition of 0.1 volume of 3 M potassium acetate and 2.5 volumes of 100% ethanol.
4. The sample was placed in ice for 40 minutes, and the precipitate formed was collected by centrifugation. (15 minutes at 10,000 rpm in an Eppendorf centrifuge)
5. Wash the pellet with 75% ethanol.
6. The pellet was dried in vacuo.
7. Add 4 μL DEPC-treated water to reconstitute the sample.

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Incubator (37℃)
4. Centrifuge

Consumables:

1. 0.2 M Tris-HCl (pH 9.5)
2. YM-10 column

Protocol

1. Add 100 μL 0.2 M Tris-HCl.
2. Incubate in a circulating water bath at 37℃ for 60 minutes.
3. Transfer reaction product to YM-10 column, centrifuge for 20 minutes at 4℃ and 10000 xg.
4. Take the supernatant and store in -30℃ freezer.

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Incubator (37℃)
4. Centrifuge

Consumables:

1. 5X Reaction buffer for S1 nuclease
2. S1 nuclease
3. Nuclease-Free Water

Protocol

1. Prepare the following reaction mixture:

   Components	Volume
   tRNA	30 μg (X μL)
   5X buffer	18 μL
   S1 nuclease	3 μL
   Nuclease-Free Water	to 90 μL (69-X μL)

2. Incubate the mixture at room temperature for 30 minutes.
3. Stop the reaction by adding 2 μL of 0.5 M EDTA and heating at 70℃ for 10 minutes.
4. Transfer reaction product to YM-10 column, centrifuge for 20 minutes at 4℃ and 10000 xg.
5. Take the supernatant and store in -30℃ freezer.

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Pyrex dish with dimensions larger than the gel
4. Glass plates
5. Spacers and 10-well comb (0.75 mm thick)
6. Casting stand and clamp
7. Electrophoresis rig
8. Power supply
9. Rotator

Consumables:

1. 40% acrylamide solution (mono:bis; 19:1 premix)
2. 3 M NaOAc (pH 5.2)
3. Urea
4. 10% ammonium persulfate (APS)
5. N,N,N',N'-tetramethylethylenediamine (TEMED)
6. Bromophenol blue
7. Methylene blue
8. Loading dye [100 mM NaOAc (pH 5.2), 7 M urea, 0.05% bromophenol blue]
9. Staining solution [500 mM NaOAc (pH 5.2), 0.06% methylene blue]

Protocol

1. Pour a 6.5% polyacrylamide gel containing 100 mM NaOAc (pH 5.2) and 8 M urea. To polymerize the gel, add 600 μL 10% APS and 60 μL TEMED per 100 mL of acrylamide solution.
2. Once polymerized, set up the gel. Use 100 mM NaOAc (pH 5.2) as a running buffer.
3. Add loading dye to the tRNA samples. Load approximately 40 pmol tRNA per lane. Do not heat samples before loading.
4. Electrophoresis for 40 minutes at 100 V, 300 A in the refrigerator.
5. Remove the spacers and then separate the plates. Carefully transfer the gel into ~ 500 mL of staining solution contained in a Pyrex dish. Place the dish on a rotator set at its lowest speed and allow the gel to stain thoroughly (~ 30 minutes).
6. Transfer the staining solution (which can be reused) into a large glass bottle using a funnel. This can be done by holding the gel against the bottom of the dish with one gloved hand while supporting the weight of the dish with the other.
7. Destain the gel with water. Several changes of water will be necessary for complete destaining. It works well to siphon the water from a corner of the dish.

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. 1X SLB or 10X SLB (sample loading buffer)
5. DNA sample
6. EtBr (Ethidium bromide)
7. RO (reverse osmosis) water

Protocol

Agarose gel preparation:

1. Adjust the amount of agarose to get the desired gel concentration (in this protocol, we will 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 into the electrophoresis tray.
6. Set the electrophoresis comb and wait for the gel to solidify.

Sample preparation:

Samples are prepared in various ways depending on their purpose.

Agarose gel running:

1. When the agarose gel solidified, take out the electrophoresis comb.
2. Pour the TBE buffer or TAE buffer into 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 5 μL 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 minutes at 100 V. Note: Voltage and time vary.

Agarose gel visualization:

1. Take the gel out of the buffer tank.
2. Soak the gel in the EtBr solution for 8 minutes and in RO water for 30 seconds.
3. Take the gel out of RO water and visualize the DNA under a UV transilluminator.

Materials

Equipment:

1. Eppendorf tubes
2. Collection tubes
3. Spin column
4. Pipette and pipette tips
5. Centrifuge
6. Scalpel
7. Dry Thermo Unit

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 (per 100 mg gel add 300 μL binding buffer).
4. Incubate at 60℃ for 2-10 minutes (or until the gel has completely dissolved).
5. Add 1.5 volumes 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 seconds at 12,000 rpm.
7. 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 seconds, 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 minutes, 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 minutes, 12,000 rpm.
2. Collect the pure sample in the eppendorf and discard the spin column.
   Keep the DNA solution in the -20℃ freezer.

Materials

Equipment:

1. Sonication
2. Centrifuge
3. His-trap
4. Nanodrop
5. Pipette and pipette tips

Consumables:

1. 1 M imidazole
2. 5 M NaCl
3. 10 mM Tris-HCl buffer (pH 7.5)
4. 1 M Tris-HCl buffer (pH 7.5)
5. Binding buffer [10 mM imidazole, 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5) buffer]
6. Elution buffer [300 mM imidazole, 0.5 M NaCl, 10 mM tris-HCl (pH 7.5) buffer]

Protocol

1. Suspend cells in 4 mL of binding buffer.
2. Disrupt the suspended cells with sonication. (Take a sample for 100 μL culture as cell suspension and mix with SDS-PAGE sample buffer.)
3. Centrifuge the disrupted cells for 10 minutes at 4℃ and 12000 rpm, then take the supernatant. (Take the sample for 100 μL culture as supernatant, and mix with SDS-PAGE sample buffer.) (Suspend the pellet with the binding buffer and take the sample for 100μL culture as pellet and mix with SDS-PAGE sample buffer.)
4. Apply to His-trap column which was washed with the binding buffer.
5. Wash the column with 5 mL of the binding buffer.
6. Elute the binding buffer protein by Elution buffer and fraction into 0.5 mL aliquots.
7. Measure protein amount by nanodrop and collect enzyme fractions.
8. Dialyze the fraction 3 times with 100 mL 10 mM Tris-HCl at 4℃.
9. Centrifuge the enzyme sample for 10 minutes at 4℃ and 12000 rpm, then transfer the supernatant to a fresh tube.
10. Determine the protein amount of the enzyme sample. (Take 1 μg and mix with SDS-PAGE sample buffer.)
11. Run SDS-PAGE. (cell suspension, supernatant, pellet, purified)

Materials

Equipment:

1. Eppendorf tubes
2. Pipette and pipette tips
3. Centrifuge
4. Vortex
5. Ice bucket
6. Empty box
7. Dry Thermo Unit
8. Glass plates
9. Spacers and 10-well comb (0.75 mm thick)
10. Casting stand and clamp
11. Electrophoresis rig
12. Power supply
13. Rotator

Consumables:

1. ddH₂O
2. 30% acrylamide
3. 1.5 M Tris-HCl (pH 8.8)
4. 0.5 M Tris-HCl (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. 1X Tank buffer
14. Isopropanol

Protocol

Resolving gel preparation

1. In a 50 mL eppendorf tube add all the components 10% gel concentration.

   Components	Volume
   ddH2O	1.36 mL
   30% acrylamide mix	1.675 mL
   1.5 M Tris-HCl (pH 8.8)	1.875 mL
   10% SDS	50 μL
   10% APS	35 μL
   TEMED	4 μL
   Total Volume	5 mL

   (Adjust the amount added by this ratio)
2. Pour the mixture in between the glasses and add isopropanol afterward.
3. Wait for 10-15 minutes or until the gel solidifies.
4. Pour out the isopropanol.

Components	Volume
ddH2O	2.1 mL
30% acrylamide mix	500 μL
0.5 M Tris-HCl (pH 6.8)	375 μL
10% SDS	30 μL
10% APS	20 μL
TEMED	3 μL
Total Volume	3.028 mL

(Adjust the amount added by this ratio)
5. Pour the mixture in between the glass plates and add the 10-well comb.
6. Wait for 10-15 minutes 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℃ for 10 minutes 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 minutes 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, and then add the CBB staining solution until it covers the gel.
3. Put the box on a shaker and shake for 30 minutes 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.

Materials

Equipment:

1. Pipette and pipette tips
2. Eppendorf tubes
3. Centrifuge
4. Flasks
5. Electroporation cuvettes (2 mm 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 500 μL of overnight culture (Escherichia coli with pKD46) to 50 mL of fresh SOB-Mg medium with 50 μL of ampicillin at 30℃.
2. Grow cells with shaking at 30℃ for 1 hour and 50 minutes.
3. Add 1 mL of 10% L-arabinose and culture for 1 hour.
4. Transfer the culture into a 50 mL centrifuge tube, and centrifuge it for 5 minutes at 4℃ 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 1 mL of chilled 10% glycerol. Transfer the cell suspension into a 1.5 mL tube and centrifuge again for 1 minute at 4℃ and 12,000 rpm (first wash).
6. Remove the supernatant using a pipette and suspend the cells with 1 mL of chilled 10% glycerol. Centrifuge again for 1 minute at 4℃ and 12,000 rpm (second wash).
7. Remove the supernatant using a pipette and suspend the cells with 1 mL of chilled 10% glycerol. Centrifuge again for 1 minute at 4℃ and 12,000 rpm (third wash).
8. Remove the supernatant using a pipette and suspend the cells with 250 μL (depends on the amount of the cell) of chilled 10% glycerol.

Electroporation:

1. Mix 50 μL of the competent cell and 5 μL DNA solution in a fresh 1.5 mL tube.
2. Transfer the mixture into a chilled electroporation cuvette (2 mm gap), and pulse (Ec 2).
3. Add 1 mL of SOC with 20 μL 10% arabinose immediately after the pulse.
4. Incubate it for 1 hour at 37℃.
5. Plate 100 μL of the culture on a selection plate, and incubate at 30℃.

Materials

Equipment:

1. Pipette and pipette tips
2. Eppendorf tubes
3. Centrifuge
4. Flasks
5. Electroporation cuvettes (2 mm gap)
6. Eletroporator

Consumables:

1. SOB
2. Chilled 10% glycerol
3. SOC
4. Selection plates
5. DNA plasmid

Protocol

Competent cell making:

1. Inoculate 1 mL of overnight culture to 50 mL of fresh SOB-Mg medium.
2. Grow cells with shaking at 37℃ for 1.5 hours (recA⁺ strain) or 1.75 hours (recA^- strain).
3. Chill the flask on ice for 5 minutes, transfer the culture into a 50 mL centrifuge tube, and centrifuge it for 5 minutes at 4℃ 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 1 mL of chilled 10% glycerol. Transfer the cell suspension into a 1.5 mL tube and centrifuge again for 1 minute at 4℃ and 12,000 rpm.
5. Remove the supernatant using a pipette and suspend the cells with 1 mL of chilled 10% glycerol. Centrifuge again for 1 minute at 4℃ and 12,000 rpm.
6. Remove the supernatant using a pipette and suspend the cells with 250 μL (depending on the amount of the cell) of chilled 10% glycerol.

Electroporation:

1. Mix 50 μL of the competent cell and DNA solution (the amount depends on the DNA plasmid) in a fresh 1.5 mL tube.
2. Transfer the mixture into a chilled electroporation cuvette (2 mm gap), and pulse (Ec 2).
3. Add 1 mL of SOC immediately after the pulse.
4. Incubate it for 1 hour at 37℃.
5. Place the culture on a selection plate, and incubate.

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 2-5 mL 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 for 30 seconds at 12,000 rpm, then discard the supernatant.
3. Add 250 μL of suspension buffer and suspend bacterial cells by vortex.
4. Add 250 μL of lysis buffer and mix gently by inverting the tube 10 times.
5. Add 350 μL of binding buffer and mix gently by inverting the tube 10 times.
6. Incubate on ice for 5 minutes.
7. Centrifuge at 4℃, 12,000 rpm for 10 minutes.
8. Place a spin column in a provided collection tube. Transfer 750 μL of supernatant to the spin column.
9. Centrifuge at room temperature, 12,000 rpm for 30 seconds and discard the flow-through.
10. Add 500 μL of washing buffer I to the spin column and centrifuge for 30 seconds. Discard the flow-through.

Materials

Equipment:

1. Eppendorf tubes
2. Collection tubes
3. Spin column
4. Pipette and pipette tips
5. Centrifuge

Consumables:

1. Binding buffer
2. Washing buffer
3. Elution buffer
4. MQ or ddH₂O

Protocol

DNA binding:

1. Add MQ or ddH₂O to DNA solution until the volume reaches 100 μL.
2. Add 500 μL of binding buffer to the solution.
3. Transfer sample to the spin column and centrifuge for 30 seconds at 12,000 rpm.
4. 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 seconds at 12,000 rpm.
2. Discard the 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 minutes at 12,000 rpm.
4. Discard the 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 minutes at 12,000 rpm.
2. Collect the pure sample in the eppendorf and discard the spin column.
3. Keep the DNA solution in -20℃ freezer.