Materials

• Suspend the sample in buffer 1 (1.5 ml sample tube)
• Add buffer 2 to the sample and mix it
• Add buffer 3 to the sample and mix it
• Centrifuge for 5 minutes at 13,000 rpm and apply the supernatants to the column
• Add buffer 4 and centrifuge for 1 minute at 13,000 rpm then throw away the filter paper
• Add buffer 5 and centrifuge for 1 minute at 13,000 rpm then throw away the filter paper
• Centrifuge again for 1 minute at 13,000 rpm
• Place the column on a new sample tube and add 100 microL distilled water
• Centrifuge for 1 minute at 6,000 rpm

Materials

• Add buffer, dNTP, primer mix, Template DNA, KOD-FX-NEO and distilled water to an Eppendorf tube and mix
• Place the PCR tubes into the PCR machine and set the program

Materials

• Add buffer, dNTP, primer mix, Template DNA, Taq polymerase and distilled water to an Eppendorf tube and mix
• Place the PCR tubes into the PCR machine and set the program

Materials

• Add all materials to a tube and mix them gently
• Incubate at 37˚C for the enzyme reaction for 15 minutes

Materials

• Set an agarose gel on an electrophoresis chamber
• Add 1X TAE buffer to the electrophoresis chamber
  Note: Do not generate bubbles under the gel
• Add 2X loading buffer to electrophoresis samples
• Apply samples on agarose gel wells
• Set an appropriate voltage (100 V) and run the electrophoresis
• Stop the electrophoresis when the BPB reaches 2/3 of the gel
• Soak the gel in EtBr (ethidium bromide) solution and dye it for 20 minutes
• Place plastic cooking wrap on the trans-illuminator and irradiate UV to the gel on the wrap
• Take photographs of the gel by using a trans-illuminator

Materials

• Add materials (DNA, H2O, CH3COONa, and Phe/Chl) to sample tube and vortex
• Centrifuge at 14,500 rpm for 10 minutes at room temperature
• Transfer the supernatant to a new sample tube
• Add 750 μL of isopropyl alcohol to the sample tube and vortex
• Centrifuge at 14,500 rpm for 10 minutes at 4°C
• Remove isopropyl alcohol gently so that the pellet will not break
• Add 1,000 μL of 75% ethanol and centrifuge at 14,500 rpm for 5 minutes at 4°C
• Dry it by aspirator for 10 minutes
• Add 25 μL of distilled H2O and mix

• Phe/Chl composition Phenol:Chloroform:Isoamyl alcohol=25:24:1

Materials

• Add BAP to vector and dephosphorize for 30 minutes at 37°C
• Run the electrophoresis with agarose gel
• Cut out the targeted band and mix with 200 μL of distilled water in a tube
• Heat the sample for 5 minutes at 65°C
• Add the same amount of phenol as the sample and vortex
• Centrifuge at 14,500 rpm for 10 minutes at room temperature and apply the supernatant to the new sample tube
• Add the same amount of Phe/Chl as the sample and vortex
• Centrifuge at 14,500 rpm for 5 minutes at room temperature and apply the supernatant to the sample tube
• Add 750 μL of Isopropyl alcohol and 50 μL of CH3COONa and centrifuge at 14,500rpm for 10 minutes at 4°C
• Rinse with 70% ethanol at 14,500 rpm 5 minutes at 4°C
• Dry it for 15 minutes

Materials

• Mix DNA sample and DNA ligase in a sample tube gently
• Ligation for 15 minutes at room temperature

Materials

• Thaw the competent cells on ice
• Add 10 μL of sample into thawed competent cells
• Cool the sample tube, which contains competent cells and DNA samples, with ice for 1 hour, then Heat shock the cells by immersion in pre-hearted water bath at 42°C for 30 seconds
• Place the tube on ice for 2 minutes to cool it down
• At a clean bench, add 1.0 ml of LB medium into the tube and suspend it
• Incubate the tube at 37°C for 35 minutes
• Harvest the cells by centrifuge
• Spread the transformed competent cells onto the agar plate and incubate it at 37°C overnight

Materials

• Cool down a cuvette for 5 minutes on ice
• Harvest the yeast cells by centrifuge at 4 °C
• Suspend the cells in 20 ml of cold 1M sorbitol and harvest by centrifuge at 4 °C (2times)
• Suspend the cells in 500μL of cold 1M sorbitol
• Mix DNA sample (5 μL) and the cell suspension (95 μL) in a cold cuvette
• Electroporate under the following conditions: 200 Ω, 25 μF and 1.5 kV
• Immediately inoculate on YPD plates containing G418
• Incubate at 30 °C for 2 days

Materials

• Scrape samples from the agar plate and inoculate them in liquid media
• Cultivate overnight (37℃, 120 rpm)

Materials

• Add samples to LB medium and cultivate (37°C, 120 rpm) until reaching a cell density of OD600=0.5
• Add IPTG (10 μL) to the medium (25 ml)
• Further cultivation for 3 hours

Materials

• Separate samples into two and harvest by centrifuge
• Add potassium phosphate buffer, then mix and remove medium completely
• Add FastBreak Cell Lysis Reagent, 10X at the ratio of FastBreak Buffer Cell Lysis Reagent,10X: Samples=1:9 and extract protein for 15 minutes at room temperature

Materials

• Harvest yeast cells by centrifuge at 3,000 rpm for 4 minutes at room temperature
• Wash cells once with potassium phosphate buffer
• Cool down the sample tube on ice for 1 minute
• Add the same amount of glass beads as the sample then add 20 μL of potassium phosphate buffer and mix
• Disrupt the cells by FastPrep 24 (MP Biochemicals) for 45 seconds at speed 4.5
• Cool down the sample tube on ice for 1 minute
• Add 80 µl of potassium phosphate buffer, cool it down and centrifuge for 5 minutes at 14,500 rpm at 4°C
• Transfer 40 µl of the supernatant and mix with 8 µl of SDS sample buffer
• Treat the sample with heat for 5 minutes at 100˚C

Materials

• Dispense cracking solution (50 μL) each into sample tubes
• Collect the sample and suspend it into cracking solution
• Incubate at 65°C for 10 minutes
• Add Phe/Chl and BPB pigment and vortex
• Centrifuge at 14,000 rpm for 5 minutes
• Apply the samples (upper layer) to agarose gel with loading dye
• Check the bands by agar gel electrophoresis

Materials

• Place stacking gel on separating gel
• Rinse the wells of gel with distilled water
• Load prepared samples into wells
• Run the electrophoresis (25 mA for 75 min)
• Check the bands with CBB stain

Materials

• Cut the gel in appropriate size
• Soak gel in buffer III and shake gently for 15 minutes
• Soak the membrane on methanol then transfer it in buffer III
• Soak Whatman papers (all in the same size) in Buffer I (2 sheets), Buffer II (1 sheet) and Buffer III (3 sheets) respectively
• Wet the surface of the blotter
• Put items in the following order: Buffer III paper, gel, membrane, Buffer II paper and Buffer I paper
• Blot at the constant current of membrane's area ×2.5 mA for 20 minutes
• Dye the membrane with Ponceau S for 5 minutes, rinse it with distilled water and scan it
• Shake and wash with PBS-TS (3 minutes ×3times)
• The membrane was incubated with anti-GST antibody containing HRP in PBS-S at room temperature for 1 hour
• Wash with PBS-T twice (5 minutes/10 minutes)
• Wash with PBS twice (5 minutes/5 minutes)
• Stain the membrane using Peroxidase Stain Kits
• Scan it

Materials

• Dissolve Tryptone (1.0 g), Yeast Extract (500 mg) and NaCl (1.0 g) in distilled water (90 ml)
• Adjust pH to 7.0 by adding 20 μL of 5M NaOH
• Fill up to 100 ml with distilled water
• Autoclave

• To make agar plate, add agar powder at the final concentration of 2 %(w/v) in Procedure 2

• Dissolve peptone (2.0 g), yeast extract (1.0 g) and glucose (2.0 g) to distilled water (90 ml)
• Fill up to 100 ml with distilled water
• Sterilize by autoclave

• To make agar plate, add agar powder (final concentration: 2.0%) at procedure 1.

Materials

• Dissolve yeast nitrogen base (6.7 g), adenine (40 mg), histidine (20 mg), leucine (60 mg), lysine (30mg), tryptophan (40mg) and methionine (20mg) into 40 ml of distilled H2O, and then fill up to 50 ml with H2O.
• Dissolve glucose (20.0 g) into 40 ml of distilled H2O, and then fill up to 50 ml with H2O.
• Sterilize each solution by autoclave.
• Mix solutions after cooling it down.

• Autoclave glucose and agar plate separately from other materials

• Prepare PCR primers (10 pmol/μl) and template plasmid (50 ng/μl)
• Prepare the PCR reaction mixture as follows:

  PCR grade water	35μL
  10×Buffer for iPCR	5μL
  2 mM dNTPs	5μL
  Primer A (10 pmol/μL)	1.5μL
  Primer B (10 pmol/μL)	1.5μL
  Plasmid Template DNA (50ng/μL)	1μL
  KOD-Plus-DNA Polymerase	1μL
  Total	50μL

  
  
• Perform PCR reaction as follows: PCR Profile for KOD-FX-NEO

  94.0°C	98.0°C	68.0°C	4.0°C
  2 minutes	10 seconds	7 minutes	Hold
  1 cycle	20 cycles		1 cycle

  
  
• [Optional] The control reaction is as follows:

  PCR grade water	35μL
  10×Buffer for iPCR	5μL
  2 mM dNTPs	5μL
  Control Primer #1（10 pmol/μl）	1.5μL
  Control Primer #2（10 pmol/μl）	1.5μL
  Control Plasmid pAK119M （50 ng/μl）	1μL
  KOD -Plus- (1 U/μl)	1μL
  Total	50μL

  
  
  

  94.0°C	98.0°C	98.0°C
  2 minutes	10 seconds	5 minutes
  1 cycle	5 cycles

　
• Add 2 μl of Dpn1 restriction enzyme (10 U/μl) to 50 μl PCR reaction from step 1.
• Gently and thoroughly mix each reaction mixture by pipetting
• Spin down the reaction mixture and incubate at 37°C for 1 hr to digest the template plasmid

（Optional） Gel electrophoresis
To confirm PCR amplification, we recommend agarose gel electrophoresis analysis. However, in the case of 5 or less cycles of amplification, it may be only possible to detect a faint band or no band at all. If only a faint or no band is detected, please proceed to the next step.

• Prepare the kination/ligation reaction mixture using a fresh tube as follows:
  

  Dpn1- treated PCR product	2μL
  PCR grade water	7μL
  Ligation high	5μL
  T4 Polynucleotide Kinase	1μL
  Total Volum	15μL

  
  
• Gently and thoroughly mix each reaction mixture by pipetting
• Centrifuge the reaction mixture and incubate each reaction at 16°C for 1 hr

Standard E. coli strains can be used as the transformation host. Both electrocompetent and chemically competent E. coli cells can be used. The following protocol is a standard protocol using chemically prepared competent E. coli cells.

• Gently thaw the competent cells （100 μｌ） on ice.
• Add 10 μｌ of reaction mixture from Step 3 to the competent cells, swirl gently and incubate for 30 min on ice.
• Incubate for 30 seconds at 42°C and then place on ice for 2 min
• Add 900 μｌ of SOC medium and incubate at 37°C for 1 hr with shaking.
• Plate appropriate volumes (10~200 μl) of transformed E. coli solution on agar plates containing the appropriate antibiotic for the plasmid vector.
• Incubate the plates at 37°C for ~16 hr.

A typical mutagenesis efficiency of this kit is greater than 80%. Therefore, analysis of 4-8 clones is typically appropriate for sequence verification.