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 20㎕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：10㎕
• Pre-cultured Bacterial cells：500㎕

Procedure

• add bacterial cells to LB medium, shake and culture so that medium turbidity gets to 0.5A (37℃120rpm)
• add IPTG (10㎕) to bacterial cell in LB medium (25㎖)
• shake and cultivate at 120rpm at 37 for 3h

Note

• measure turbidity by OD600

Transformation (E.coli)

Materials

• DNA Sample：10㎕
• Competent cell：20㎕
• 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 10㎕ 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 37℃ for 35 minutes
• harvest the cells by centrifuge.
• Seed the transformed competent cells onto the agar medium
• incubate the plate at 37℃ 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℃ 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, 50㎕ each, into tubes
• collect the sample and suspend it into cracking solution
• incubate at 65℃ 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}：5㎕
• 1.0% agarose gel
• 2×Loading Buffer：5㎕

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/㎕ 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)：1㎕

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