Protocol

LB Agar

Materials

Agar powder	final concentration: 1.5 %(w/v)
LB medium

Procedure

• Add agar powder (6.0 g) to LB medium (400 ml)
• Dissolve it by autoclaving
• Stir up with magnetic stirrer
• Cool it down to the room temperature in order to make it to gel form

YPD Broth/YPD Medium

Materials

Peptone	final concentration: 2%(w/v)
Yeast extract	final concentration: 1%(w/v)
Glucose	final concentration: 2%(w/v)

Procedure

• 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

Note

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

Main Culture

Materials

LB medium	100ml
IPTG	10μL
Sample	500μL

Procedure

• Add samples to LB medium, cultivate at 37°C in shaken culture (120 rpm) until reaching a cell density of OD600 0.5A
• Add IPTG (10 μL) to the sample (25 ml)
• Cultivate at 37°C in shaken culture (120 rpm) for 3 hours

Note

Transformation (E.coli)

Materials

Sample	10μL
Competent cell	20μL

Procedure

• Thaw the competent cells on ice
• Add 10 μL of sample into thawed competent cells
• Cool an Eppendorf tube, which contains competent cells and samples, with ice for one hour, then Heat shock the cells by immersion in pre-hearted water bath at 41°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
• Seed the transformed competent cells onto the agar plate and incubate it at 37°C overnight

Pre-culture

Materials

Sample
Medium	20ml

Procedure

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

Protein Extraction (E.coli)

Materials

Sample
Fast Break Cell Lysis Reagent, 10X (Promega)
50mM potassium phosphate buffer (=pH6.8)
SDS sample buffer

Procedure

• 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

Colony Sweep

Materials

Sample

Phe-Chl

Cracking solution

Procedure

• Dispense cracking solution (50 μL) each into Eppendorf 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
• Check the bands by agar gel electrophoresis

AGE

Materials

{Sample}	5μL
Agarose gel
2X Loading Buffer Triple Dye (NIPPON GENE)	5μL
1X TAE Buffer

Procedure

• 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 and run the electrophoresis
• Stop the electrophoresis when the BPB reaches 2/3 of the gel
• Soak the gel in 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

PCR

Materials

Buffer for KOD-FX-NEO
dNTP	20μL
Primer mix	1μL
KOD-FX-NEO	2μL
H2O	26.5μL
Total	50μL

Procedure

• Bring the volume up forward primer to 100 pmol/μL with sterile dH2O
• Add 10 μL of this primer solution and 80 μL of H2O into another tube
  Make 10 times dilution
• Use 1 μL primer mix for PCR
  Reaction composition is below

Ligation

Materials

DNA sample (cut out from the gel)
Distilled water	5μL
DNA ligase	5μL

Procedure

• Add DNA sample, distilled water and DNA ligase into a micro test tube and vortex
• Ligation for 5minutes at room temperature

Western Blotting

Materials

BufferⅠ
BufferⅡ
BufferⅢ
Distilled water	2ml
PBS
PBS-S
PBS-T
PBS-TS
PonceauS
PVDF membrane	1 sheet
Whatman paper	6 sheets
Hybridization bag	1
Peroxidase Stain Kit	one drop for each
antiglutathione S - transferase (和光純薬工業株式会社製)	1μL

Procedure

• 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

Reagent

• 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