Protocol

LB Broth/ LB Medium

Materials

Tryptone final concentration	1%(w/v)
Yeast Extract final concentration	0.5%(w/v)
Sodium Chloride F.W.=58.44 final concentration	1%(w/v)
5M Sodium Hydroxide solution

Procedure

• Dissolve tryptone (1.0g), Yeast Extract (500mg) and Sodium Chloride (1.0g) in distilled water(90ml)
• Adjust pH to 7.0 by adding 20μL of 5M sodium chloride
• Dilute solution with distilled water and bring volume to 100ml
• Autoclave

Note

• Add antibiotics to medium at 1/1000

LB Agar

Materials

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

Procedure

• Add agar powder (6.0g) to LB medium (400ml)
• 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)
L Glucose final concentration	2%(w/v)

Procedure

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

Note

• Kanamycin at 20(μg/ml)

Main Culture

Materials

LB medium	100cc
IPTG	10μL
Pre-cultured Bacterial cells	500μL

Procedure

• Add bacterial cells to LB medium, shake and culture so that medium turbidity gets to 0.5A (37°C120rpm)
• Add IPTG (10μL) to bacterial cell in LB medium (25ml)
• Shake and cultivate at 120rpm at 37°C for 3 hours

Note

• Measure turbidity by OD600

Transformation (E.coli)

Materials

DNA Sample	10μL
Competent cell	20μL
LB agar plate with Amp	same number of plates as the kind of DNA samples
LB medium

Procedure

• Thaw the competent cells on ice
• Add 10μL of DNA sample into thawed competent cells
• Cool the tube, which contains competent cells and DNA 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 1.0ml 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 medium
• Incubate the plate at 37°C overnight

Pre-culture

Materials

• Bacterial cell (negative control: bacterial cells which have been transferred from empty vector) 20ml
• Medium (with and without antibiotics. Seed bacterial cells on the one without antibiotics as a negative control) 20ml

Procedure

• Scrape bacterial cells from the agar plate and incubate them on a Medium
• Cultivate it in a shake-flask at 37°C overnight

Protein Extraction (E.coli)

Materials

• Bacterial cells 100ml
• Fast Break Buffer
• 50mM potassium phosphate buffer (=pH6.8)
• SDS sample buffer

Procedure

• Separate bacterial cells into two and harvest by centrifuge
• Add potassium phosphate buffer, then mix and remove medium completely
• Add Fast Break Buffer at the ratio of Fast Break Buffer: Bacterial cells=1:9 and extract protein (R.T15min)

Rapid Screening for the Detection of Recombinant Plasmids

Materials

• DNA sample
• Phe-Chl
• Cracking solution

Procedure

• Dispense cracking solution, 50μL each, into tubes
• Collect the sample and suspend it into cracking solution
• Incubate at 65°C for 10 minutes
• Add Phe-Chl and a BPB pigment and vortex it
• Centrifuge it
• Check the band by agar gel electrophoresis

AGE

Materials

• {Sample} 5μL
• 1.0% agarose gel
• 2×Loading Buffer 5μL

Procedure

• Set the 1.0%agarose gel on the electrophoresis chamber
• Add 1×Loading Buffer into the electrophoresis chamber
  Note: do not generate bubbles under the gel
• Add 2×Loading Buffer into the electrophoresis sample
• Apply sample on the agarose gel well
• Electrophoresis
• Stop 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 100pmol/μL with sterile dH2O .
• Add 10μL of this primer solution and 80μL of H2O into another tube.
  Make 10 times dilution.
• Use 1μL 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 (R.T for 5minutes)

Western Blotting

Materials

• BufferⅠ appropriate amount
• BufferⅡ appropriate amount
• BufferⅢ appropriate amount
• Distilled water 2ml
• PBS appropriate amount
• PBS-S appropriate amount
• PBS-T appropriate amount
• PBS-TS appropriate amount
• PonceauS appropriate amount
• 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 buffer 3 and gel then shake it gently
• Soak the membrane on ethanol then soak it in buffer 3 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 PonceauS for five minutes rinse it with distilled water and scan it
• Shake and wash with PBS-TS (3 minutes ×3times)
• Put the membrane in a hybridization bag add PBS-S antiglutathione S - transferase and shake it (R.T. one hour)
• Shake and wash with PBS-T twice (5min/10min)
• Shake and wash with PBS twice (5min/5min)
• Add one drip of 3 Peroxidase Stain Kits and distilled water
• Scan it

Reagent

• BufferⅠ: bring to 300ml with Tris base 10.9g,MetOH60ml/H2O
• BufferⅡ: bring to 300ml with Tris base 0.9g,MetOH60ml/H2O
• BufferⅢ: bring to 300ml 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