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 (500㎎) and Sodium Chloride (1.0g) in distilled water(90㎖)
• Adjust pH to 7.0 by adding 20μL of 5M sodium chloride
• Dilute solution with distilled water and bring volume to 100㎖
• 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 (400㎖)
• 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 (90㎖)
• Dilute solution with distilled water and bring up volume to 100㎖
• Sterilize by autoclave

Note

• Kanamycin at 20(㎍/㎖)

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℃120rpm)
• Add IPTG (10μL) to bacterial cell in LB medium (25㎖)
• Shake and cultivate at 120rpm at 37ºC 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
• Ice

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 41ºC 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 37ºC for 35 minutes
• Harvest the cells by centrifuge.
• Seed the transformed competent cells onto the agar medium
• Incubate the plate at 37ºC overnight

Pre-culture

Materials

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

Procedure

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

Protein Extraction (E.coli)

Materials

• Bacterial cells：100cc
• 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 65ºC 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：2㎖
• 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