Team:Osaka/Protocols

Protocols

• Preparation of Competent cells and Heat-shock Transformation
  • Preparation

1. SOB medium 1L (-> final conc.)
   • Bacto Tryptone 20g (-> 2.0%)
   • Bacto Yeast Extract 5g (-> 0.5%)
   • 5M NaCl 2ml (-> 10mM)
   • 2M KCl 1.25ml (-> 2.5mM)
   • Add miliQ water 990ml to all miture and sterilize by autoclave. Before using, add sterilized Mg2 solution (1M MgSO4, MgCl2) 10 ml.
2. SOC medium
   • Add sterilized 2M glucose 1 / 100 volume to SOB medium, and sterilize by filtration using 0.22 um microfilter. Store at 4℃.
3. Tranformation buffer, TB 1L (-> final conc.)
   • PIPES 3.0g (-> 10mM)
   • CaCl2 2H2O 2.2g (-> 15mM)
   • KCl 18.6g (-> 250mM)
   • After this mixture is suspended in about 950ml sterilized water, control the pH to 6.7-6.8 by 5N KOH (5M KOH). In low pH, this mixture cannot dissolve. Next, add MnCl2 4H2O (10.9g) to the solution, so as to be final concentration 55mM. Ajust it to 1L and sterilize by filtration using 0.22 um microfilter. Store at 4℃.
4. Liquid nitrogen
5. DMSO (dimethyl sulfoxide)

• • Preparation of competent cell

1. Streak E. coli appropriate strain on LB plate. Incuabte at 37℃ overnight.
2. Inoculate single colony (1-3mm in diameter) into 1ml SOB medium and incubate at 37℃ overnight. After that, inoculate overight culture into SOB 250ml provided in 3L conical flask.
3. Incubate at 18℃ for 19-50 hour with shaking (>200rpm).
4. If OD600 reach at 0.4-1.5 (anyphase may be possible to use), immediately put cell culture flask onto ice water for 10min.
5. Decant cell culture to 500ml centrifuge tube (or 250ml tube X2) and centrifuge in 3000rpm for 15min at 4℃.
6. Remove soup, suspend ppt by ice-cold TB 80ml, put it onto ice water for 10min.
7. Centrifuge in 3000rpm for 15min at 4℃.
8. Suspend ppt by 20ml ice-cold TB and add 1.5ml DMSO (final conc. 7%). Moreover, put on ice water for 10min.
9. Dispence 0.1-0.5ml each to 1.5ml tube and immediately transfer all tubes into liquid nitrogen. Cold shock is needed for competent cell.
10. Store at -80℃ until to use.

• • Transfomation method

1. Melt competent cell taken from freezer in hand and put on ice.
2. After dispencing 10-50μl each competent cell, add 1-20μl DNA. Put on ice for 30min.
3. Heat shock at 42℃ in water bath for 30sec and immediately put onto ice for 2min.
4. Add 40-200μl SOC medium and incubate at 37℃ for 1hour with shaking.
5. Plate the culture onto LB with appropriate antibiotics, for example ampicillin, kanamycin,chloramphenicol, etc. Incubate at 37℃ overnight.

• Plasmid DNA miniprep
  • We use GenElute™ Plasmid Miniprep Kit (SIGMA-ALDRICH) and refer to this accessory protocol (Japanese) .

• 3A BioBrick assembly
  • We use BioBrick™ Assembly Kit (NEB) and refer to OpenWetWare protocol.

• Extraction of genome DNA for PCR cloning

1. E. coli or S. cerevisiae are cultured overnigt.
2. Suspend about 10⁵ cells into sterilized miliQ-water and place in boiling water for 5 min. When treating S. cerevisiae, wash cells once by PBS buffer. PBS buffer is 25mM sodium phosphate pH 7.0-7.2, 125mM NaCl₂.
3. Chill on ice for 2 min, then spin down for 3 min by microcentrifuge at 13,000 g.
4. Transfer the supernatant to a fresh tube. Use 2.5-10ul in one PCR.
   • Reference: M. J. McPherson et al., PCR: a practical approach, 1991.

• Gene cloning
  • We perfome gene cloning as below.

1. PCR.
   • We use TaKaRa Ex Taq® for amplifying DNA and extracted genome DNA and gifted plasmids as template.
   • General cycle condition is 25 cycle; 94℃, 30sec, 55℃, 30sec, 72℃, 1min.
   • If non-specific products are heavy, annealing temperature (55℃) is changed higher.
   • If the length of gene is long or short, elongnation time (72℃ reaction time) is changed in that case.
2. Purification of PCR product.
   • We use QIAquick PCR Purification Kit for purification.
3. Restriction Digest.
   • Purified PCR products are digested by proper restriction enzymes; two of EcoRI, XbaI, SpeI and PstI.
4. Ligation.
   • We use T4 DNA Ligase for ligation.
   • Refer to 3A BioBrick assembly.
5. Transfomation.
   • Refer to Preparation of Competent cells and Heat-shock Transformation.
6. Selection and Cultivation.
   • We select plasmid-introduced colony by discrimination of marker gene (rfp) and cultivate it in LB liquid medium at 37℃ overnight.
7. Miniprep and Check
   • Refer to Plasmid DNA miniprep.
   • Plasmid Check is performed by restriction digest.
8. 　Sequence.
   • Obtained Biobrick parts are sequenced.

• PCR site-directed mutagenesis (Overlap extension PCR)
  • We refer to introduction of site-specific mutagenesis (Japanese) .

1. Design two mutagnesis primers, which are sense and antisense to target sequence.
2. First PCR. This is normal PCR as noted avobe with mutagenesis primer(sense or antisense) and flanking primer(antisense or sense). Reduce template to prevent comtamination of wild type template for second PCR.
3. Second PCR. This PCR introduces site-specific mutagenesis to target gene. Use first PCR product as template. To begin with, run PCR in 2 cycle without the ends of primers. Next, add those primers and perform normally PCR.

• Congo red plate assay
  • Preparation

1. 0.1% congo red solution
   • Congo red (KISHIDA) is water-soluble.
2. 1% CMC medium
   • CMC medium is LB medium containing 1% CMC (carboxymethylcellulose, CMC).
3. 1M NaCl
4. 5% acetic acid

• • Plate assay

1. Spread 0.1% Congo red solution 5ml onto CMC plate entirely and stain it for 30min.
2. Discard fully-reacted solution and rinse the plate surface by distilled water.
3. Add 1M NaCl 5ml and leave for 5min.
4. Discard NaCl solution and rinse plate surface by distilled water.
5. Add 5% acetic acid and leave for 5min.
6. Observe and record the halo size.