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

• 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 20μL 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

Materials

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

• To make agar plate, add agar powder (final concentration: 1.5%) at procedure 1.

Materials

• Add samples to LB medium and cultivate (37℃, 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

• 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

• Scrape samples from the agar plate and inoculate them on medium
• Cultivate at 37°C in a shaken culture overnight

Materials

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

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

• 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 (100v) and run the electrophoresis
• Stop the electrophoresis when the BPB reaches 2/3 of the gel
• Soak the gel in EtBr (ethidium bromide) 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 DNA sample, distilled water and DNA ligase into a micro test tube and vortex
• Ligation for 5minutes at room temperature

Materials

• Cut the gel in appropriate size
• Add BufferⅢ and gel then shake it gently
• Soak the membrane on ethanol then soak it in BufferⅢ and percolate
• 2, 1, 3 Whatman papers (all in the same size) on BufferⅠ, BufferⅡ, BufferⅢ respectively. wet the surface of the blotter
• Blot at the constant current of membrane's area ×2.5 mA
• 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 ×3 times)
• Put the membrane in a hybridization bag add PBS-S antiglutathione S-transferase and shake it for one hour at room temperature
• Shake and wash with PBS-T twice (5 min/10 min)
• Shake and wash with PBS twice (5 min/5 min)
• Add one drip of 3 Peroxidase Stain Kits and distilled water
• Scan it

• BufferⅠ: bring to 300 ml with Tris base 10.9g, MetOH60ml/H2O
• BufferⅡ: bring to 300 ml with Tris base 0.9g, MetOH60ml/H2O
• BufferⅢ: bring to 300 ml with Tris base 0.91g, Boric acid10.5mg, MetOH60ml/H2O
• PBS-S: PBS with 1% SkimMilk
• PBS-T: PBS with 0.05% Tween20
• PBS-TS: PBS with 0.05% Tween20+1% SkimMilk