From 2013.igem.org

Revision as of 05:57, 23 September 2013

Material

Parts

<groupparts>iGEM013 Kyoto</groupparts>

Strains

E.coli(K12)

• JM109
  
• XL10-gold
  
• BL21(DE3)pLysS
  
• JW0588
  

Protocol

PCR

• Mix the following.
  

• Put samples into Thermal Cyclers and run the following steps.

• Agarose Gel Electrophoresis for confirmation.
  
  

PCR: ToYoBo Quick Taq HS DyeMix

• Mix the following.
  

• Put samples into Thermal Cyclers and run the following steps.

• Agarose Gel Electrophoresis for confirmation.
  

RNA Extraction

Use　ISOGEN－LS（NIPPON　GENE,311-02621

• Add 1mL ISOGEN-LS to sample and vortex.
  
• Store for 5min on ice.
  
• Add 250µL chloroform and shake vigorously for 15 sec.
  
• Store for 3min. on ice.
  
• Centrifuge 17400xg for 10min. at 4°C.
  
• Transfer aqueous phase to another tube and add 0.8 volume isopropanol.
  
• Store for 10min. on ice.
  
• Centrifuge 17400xg for 10min. at 4°C.
  
• Discard the supernatant.
  
• Add 800µL 80% ethanol and vortex.
  
• Centrifuge 7500xg for 5min. at 4°C.
  
• Discard the supernatant.
  
• Dry briefly.
  
• Dissolve in nuclease-free water.
  

Making Competent Cells

• Streak E.coli cells on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)
  
• Allow cells to grow at 37°C overnight
  
• Place one colony in 10 mL LB media (+antibiotic selection if necessary), grow overnight at 37°C
  
• Take 2 ml LB media and save for blank. Transfer 5 mL overnight DH5a culture into 500 mL LB media in 3 L flask
  
• Allow cell to grow at 37°C (250 rpm), until OD600= 0.4 (~2-3 hours)
  
• Transfer cells to 2 centrifuge bottles (250 mL), and place cells on ice for 30 mins
  
• Centrifuge cells in Sorval GSA rotor at 4°C for 10 mins at 3,000 g.
  
• Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in a cold room
  
• Pour off media and resuspend cells in 12 mL of cold TB.
  
• Centrifuge cells using Sorval RT6000B rotor at 4°C for 10 mins at 3,000 g (2500 rpm)
  
• Pour supernatant and resuspend cells (by pipetting) in 5 mL of cold TB and 350 µL of DMSO. Transfer 20 µL to 1.5 mL　tube
  
• Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80°C can be used for transformation for up to ~6 months
  

Miniprep

Use LaboPass Plasmid Mini Purification Kit.

• Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 4mL Plusgrow medium containing the appropriate selective antibiotic.
  
• Incubate at 160 rpm for 8 h at 37 °C with vigorous shaking.
  
• Pellet the bacterial culture by centrifugation for 1 min at 15,000 g in a tabletop centrifuge.
  
• Discard the supernatant as much as possible.
  
• Resuspend pelleted bacterial cells in 250 µL of Buffer S1.
  
• Transfer the suspension to a new 1.5 mL tube.
  
• Add 250 µL of Buffer S2 and mix by inverting the tube 4 times (do not vortex).
  
• Add 350 µL of Buffer S3 and immediately mix by inverting the tube 4-6 times (do not vortex).
  
• Centrifuge for 10min.
  
• Transfer carefully the supernatant to a spin column and centrufuge for 1 min.
  
• Remove the spin column, discard the flowthrough, and re-insert the spin column to the collection tube.
  
• Apply the 750 µL of Buffer PW and centrifuge for 1 min.
  
• Remove the spin column, discard the flowthrough, and re-insert the spin column to the collection tube.
  
• Discard the flowthrough, and centrifuge for an additional 1 min to remove residual wash buffer.
  
• Transfer the spin column to a new 1.5 mL tube.
  
• Add 30 µL of MilliQ, let stand for 1 min, and centrifuge for 1 min.
  

Ethanol Precipitation

Use Ethachinmate (NIPPON GENE、312-01791).

• Add 3.3 µL of 3M Sodium Acetate (attached with Ethachinmate) into 100µL of DNA solution.
  
• Add 1µL of Ethachinmate.
  
• Vortex.
  
• Add ethanol, 200-250µL.
  
• Vortex.
  
• Centrifuge at 12000xg for 5min.
  
• Precipitation.
  

Electrophoresis

• Prepare 200mL of a 1.0% agarose solution:
  
• Measure 2.0g agarose into a beaker.
  
• Add 200mL 1xTAE buffer.
  
• Wrap the top of the beaker with plastic wrap.
  
• Punch a hole through the wrap with a pipette tip (To let out steam).
  
• Dissolve the agarose by heating in microwave and swirling without boiling.
  
• Allow the agarose to cool.
  
• Pour the agarose solution into a gel tray on a gel maker.
  
• If there is air bubbles, pushing them with a pipette tip.
  
• Place comb in the maker.
  
• Cover the maker with a plastic wrap.
  
• Let stand for about 45min.
  
• Remove the comb carefully.
  
• Store in the Tupperware in the refrigerator.
  
• Place the tray in electrophoresis chamber.
  
• Cover the tray with 1xTAE buffer.
  
• To prepare samples for electrophoresis, add 1µL of 10x Loading Buffer for every 9µL of DNA solution and mix well.
  
• Load 6µL of the DNA solution per well.
  
• Electrophoresis at 100V for about 30min until Loading Buffer have migrated approximately three-quarters of the gel.
  
• Stain the gel in 0.5µg/mL ethidium bromide for 20-30min.
  
• Place a plastic wrap on the transilluminator in the cabinet of Printgraph.
  
• Place the gel on the transilluminator.
  
• Turn on the transilluminator and confirm the position of the gel.
  
• Shoot the picture.
  
• Turn off the transilluminator.
  
• Dispose of the gel.
  

PCR Purification and Gel Extraction

まだ書かれていない Use Wizard SV Gel and PCR Clean-Up System by Promega.

• Combine 1 part sample (PCR product) with one part Membrane Binding Solution (e.g. 50µl sample+ 50µl).
  
• Apply the solution to the column, and let stand for 1min.
  
• Centrifuge for 1min at 13000rpm. Discard the flow-through.
  
• Add 700µl Membrane Wash Solution.
  
• Centrifuge for 1min and discard the through.
  
• Add 500µl Membrane Wash Solution.
  
• Centrifuge for 5min and discard the through.
  
• Place the column in a clean tube.
  
• Add 60µl MilliQ to the center of each column, let stand for 1min.
  
• Centrifuge for 1min at 13000rpm.
  
• Discard the column.
  

qRT-PCR

Use QuantiTect SYBR green PCR kit Cat. No. 204143 by QIAGEN

• Dilute primer to 1.5µM.
  
• Dilute RT products.
  
• Mix the following;
  

• Mix the reaction mix thoroughly, and dispense 36µL into 96 wells plate.
  
• Add primer set 9µL.
  
• Mix by inverting and voltex.
  
• Dispense 10µL into 384 wells plate and centrifuge.
  
• Let stand for 2min at 50°C and for 15min for 95°C.
  
• 40 cycles for 15sec at 95°C, for 30sec at 60°C, and for 1min at 72°C.
  
• Let stand for 15sec at 95°C.
  

Ligation

• Make 2µL of Mixture (the vector and the insert at 1 : 5-10) and Control (only the vector).
  
• Add 5µL Ligation High, 1µL T4 Kinase, and 7µL MilliQ to create a solution.
  
• Incubate at 16°C for 30 min. If the colonies of E.coli transformed with the Control
  

Restriction Enzyme Digestion

Use EcoRI, XbaI, SpeI, PstI, (NEB)

• Mix the following.

• Let stand for 2h at 37°C
  

Media

M9 medium

• Stir Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g and NH4Cl 1 g with water 500 mL.
  
• If you make M9 plates, add agar 15 g.
  
• After autoclave, add 1 mL filter sterilized 1 M MgSO4, 5.6 mL 2 M glucose, 0.1 mL 1 M CaCl2, and 1 mL 1 % thiamine-HCl.
  

If you need, add 10 mL filter sterilized 20 % casamino acid.
LB medium

• Stir BactoTryptone 2 g, Bacto-yeast extract 1 g, NaCl 1 g and 1M NaOH 200 µL with water 200mL.
  
• If you make LB plates, add agar 2 g.
  
• Autoclave.
  

SOB medium

• Stir BactoTryptone 20 g and Bacto-yeast extract 5 g with water.
  
• Add 2 mL 5 M NaCl and 840 µL 3 M KCl and add water up to 1 L.
  
• After autoclave, add 10 mL filter sterilized 1 M MgSO4 and 1 M MgCl2.
  

SOC medium

• Add 2 M glucose 1 mL to 100 mL SOB.
  

Plusgrow Medium

• Stir plusgrow 20 g and with water 500 mL.
  
• Autoclave.
  

Buffer TB

• Stir 0.6g PIPES and 30mg CaCl2 with 10 mL water.
  
• Add 2.5mL 2M KCl.
  
• Add KOH and adjust pH 6.7.
  
• Add 0.218g MnCl2・4H2O.
  
• Add water up to 20 mL.
  
• Filter sterilize.
  

Solubilization of Antibiotics

• Mix the following (Final concentration is 50mg/mL).
  

Ampicillin

Kanamycin

• Dispense 1.1mL of the solution into 1.5mL tubes.
  
• Store in the -20°C freezer.
  

Transformation

• Unfreeze conpetent cells on ice.
  
• Dry a plate by letting the plate upside down and partly open in incubator.
  
• Add 1~20µL DNA solution and 10~50µL competent cells to 1.5mL tube, let stand for 30min on ice. If few colonies are observed, increase the amount of the competent cells or DNA, but make the amount of DNA not to get over that of the competent cells.
  
• Heatshock for 45s at 42°C.
  
• Let stand for 2min on ice.
  
• Culture for 1h in preculture medium (LB or SOC medium), and plate by using spreader. Do not heat spreader too much because E.coli will dead for heat.
  
• Culture overnight at 37°C.
  

Sequence

Use Big Dye Terminator 3.1（ABI）

• Mix the following
  

• Let stand for 1min at 96°C.
  
• 35 cycles for 5s at 98°C, for 5s 50°C, and for 2.5min at 68°C.
  
• Add 25µL 100% ethanol and 1µL NAOAC
  

Golden Gate Assembly

• Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces to a 15 µl total volume assembly reaction mixture as follows:

NOTE: It is essential to use a High Concentration Ligase

BsaI is only 10% active at 37 C without the addition of BSA.

• Perform the assembly reaction in a thermocycler as follows:

either (following Engler 2009):
3 min @ 37 C }
4 min @ 16 C } 25 cycles
5 min @ 50 C }
5 min @ 80 C } 1 cycle

• Transform 5 µl of the assembly reaction into 100 µl of competent E. coli and/or run a diagnostic agarose gel to check for successful assembly.