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Contents 1 Protocol 1.1 PCR 1.2 RNA Extraction 1.3 Making Competent cells 1.4 Miniprep 1.5 Ethanol Precipitation 1.6 Electrophoresis 1.7 PCR Purification 1.8 qRT-PCR 1.9 Ligation 1.10 Restrictive Digestion 1.11 Media 1.12 Buffer TB 1.13 Solubilization of Antibiotics 1.14 Transformation 1.15 Sequence 1.16 Golden Gate Assembly Protocol 2 Material 2.1 Strains 2.1.1 E.coli

Protocol

PCR

PCR: ToYoBo KOD FX or ToYoBo KOD PLUS

• Dilute template DNA. If the concentration of DNA is 2-100ng/µL, transfer 1µL to a clean tube and add 99µL MilliQ.
  
• Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µL to a clean tube and add 99µL MilliQ.
  
• Mix the following.
  

For use of KOD plus ver2

25mM MgSO4	3µL
2mM dNTPs	5µL
10xBuffer for KOD plus ver.2	5µL
Template DNA (5ng/µL)	5µL
Primer Forward (10µM)	1.5µL
Primer Reverse (10µM)	1.5µL
KOD plus ver.2	1µL
MilliQ	28µL
Total	50µL

For use of KOD FX

2mM dNTPs	10µL
2xBuffer for KOD FX	25µL
Template DNA	5µL
Primer Forward (10µM)	1.5µL
Primer Reverse (10µM)	1.5µL
KOD FX	1µL
MilliQ	6µL
Total	50µL

• Let stand for 2min at 94℃.
  
• 25-40 cycles for 10s at 98℃, for 30s at Tm-5℃, and for 1min (1min for 1kb) at 68℃ (Tm is temperature at which primer will dissolve).
  
• Agarose Gel Electrophoresis for confirmation.
  
  

PCR: Takara Ex taq

• Mix the following (Do on PCR Bench).
  

10x PCR buffer (TAKARA)	40µL
2.5mM dNTP	8µL
Primer-1 (10pmol/µL)	8µL
Primer-2 (10pmol/µL)	8µL
Ex Taq HS (TAKARA)	1.6µL
MilliQ	334µL (to total 400µL)
Total	400µL

• Dispense 25µL to 15 tubes.
  
• Pick a single colony and transfer it to each tube.
  
• Suspend the colony.
  
• Let stand for 10min at 90℃.
  
• 35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
  
• Let stand for 4min at 72℃.
  
• Add 5mL Loading Buffer to the tubes.
  
• Agalose Gel Electrophoresis for confirmation.
  
• Negative Control: Use nothing.
  
• Positive Control: Use a colony that will yield a product with these primers.
  

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℃.
  
• Transfer aqueous phase to another tube and add 0.8 volume isopropanol.
  
• Store for 10min. on ice.
  
• Centrifuge 17400xg for 10min. at 4℃.
  
• Discard the supernatant.
  
• Add 800µL 80% ethanol and vortex.
  
• Centrifuge 7500xg for 5min. at 4℃.
  
• 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℃ overnight
  
• Place one colony in 10 mL LB media (+antibiotic selection if necessary), grow overnight at 37℃
  
• 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℃ (250 rpm), until OD600= 0.4 (~2-3 hours)
  
• Transfer cells to 2 centrifuge bottles (250 mL), and place cells on ice for 20 mins
  
• Centrifuge cells in Sorval GSA rotor at 4℃ 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 the cold room
  
• Pour off media and resuspend cells in 30 mL of cold 0.1 M CaCl2. Transfer the suspended cells into 50 mL polypropylene falcon tubes, and incubate on ice for 30 mins
  
• Centrifuge cells using Sorval RT6000B rotor at 4℃ for 10 mins at 3,000 g (2500 rpm)
  
• Pour supernatant and resuspend cells (by pipetting) in 8 mL cold 0.1M CaCl2 containing 15% glycerol. Transfer 140 mL into (1.5 mL)
  
• Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80℃ can be used for transformation for up to ~6 months
  

Miniprep

Use QIAprep Spin Miniprep Kit Cat. No. 27104 by QIAGEN

• Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 3mL LB medium containing the appropriate selective antibiotic.
  
• Incubate at 170rpm for 8h at 37℃ with vigorous shaking.
  
• Transfer a half of the culture to a tube.
  
• Harvest the bacterial cells by centrifugation at 14,000g for 1min at 4℃. Remove the medium by decanting.
  
• Transfer the half of the culture to same tube and harvest as same. Remove the medium by pipetting.
  
• Resuspend pelleted bacterial cells in 250µL Buffer P1 and mix thoroughly by pipeting.
  
• Add 250µL Buffer P2 and mix thoroughly by inverting the tube gently 4-6 times.
  
• Add 350µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  
• Centrifuge for 10min at 14,000g at 4℃.
  
• Apply the supernatants from step 10 to the QIAprep spin column by pipetting.
  
• Centrifuge for 10s in a table-top microcentrifuge. Discard the flow-through.
  
• Wash the QIAprep spin column by adding 0.5mL Buffer PB and centrifuging for 10s in a table-top microcentrifuge. Discard the flow-through.
  
• Wash QIAprep spin column by adding 0.65mL Buffer PeE and centrifuging for 10s in a table-top microcentrifuge.
  
• Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.
  
• Place the QIAprep column in a clean tube. To elute DNA, add 50µL water to the center of each QIAprep spin column, let stand for 1min, and centrifuge for 1min.
  
• Discard the QIAprep spin column.
  
• Measure the concentration of DNA by using eppendorf BioPhotometer plus.
  
• Restriction Digestion.
  
• Agarose Gel Electrophoresis for Confirmation.
  

Use Wizard Plus SV Minipreps DNA Purification System by Promega

• Harvest 1–5ml (high-copy-number plasmid) or 10ml (low-copy-number plasmid) of bacterial culture by centrifugation for 5 minutes at 10,000 x g in a tabletop centrifuge. Pour off the supernatant and blot the inverted tube on a paper towel to remove excess media.
  
• Add 250µl of Cell Resuspension Solution and completely resuspend the cell pellet by vortexing or pipetting. It is essential to thoroughly resuspend the cells. If they are not already in a microcentrifuge tube, transfer the resuspended cells to a sterile 1.5ml microcentrifuge tube(s).
  
• Add 250µl of Cell Lysis Solution and mix by inverting the tube 4 times (do not vortex). Incubate until the cell suspension clears (approximately 1–5 minutes).
  
• Add 10µl of Alkaline Protease Solution and mix by inverting the tube 4 times. Incubate for 5 minutes at room temperature.
  
• Add 350µl of Neutralization Solution and immediately mix by inverting the tube 4 times (do not vortex).
  
• Centrifuge the bacterial lysate at maximum speed (around 14,000 × g) in a microcentrifuge for 10 minutes at room temperature.
  
• Transfer the cleared lysate (approximately 850µl, Section 3.B, Step 6) to the prepared Spin Column by decanting. Avoid disturbing or transferring any of the white precipitate with the supernatant.
  
• Centrifuge the supernatant at maximum speed in a microcentrifuge for 1 minute at room temperature. Remove the Spin Column from the tube and discard the flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection Tube.
  
• Add 750µl of Column Wash Solution, previously diluted with 95% ethanol, to the Spin Column.
  
• Centrifuge at maximum speed in a microcentrifuge for 1 minute at room temperature. Remove the Spin Column from the tube and discard the flowthrough. Reinsert the Spin Column into the Collection Tube.
  
• Repeat the wash procedure using 250µl of Column Wash Solution.
  
• Centrifuge at maximum speed in a microcentrifuge for 2 minutes at room temperature.om temperature.
  
• Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column. If the Spin Column has Column Wash Solution associated with it, centrifuge again for 1 minute at maximum speed.
  
• Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube.
  
• Elute the plasmid DNA by adding 100µl of Nuclease-Free Water to the Spin Column. Centrifuge at maximum speed for 1 minute at room temperature in microcentrifuge.
  
• After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube and discard the Spin Column.
  
• DNA is stable in water without addition of a buffer if stored at –20°C or below. DNA is stable at 4°C in TE buffer. To store the DNA in TE buffer, add 11µl of 10X TE buffer to the 100µl of eluted DNA. Do not add TE buffer if the DNA is to be used for automated fluorescent sequencing.
  
• Cap the microcentrifuge tube and store the purified plasmid DNA at –20°C or below.
  

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 6x Loading Buffer for every 5µL 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.
  
• Rinse the gel with MilliQ.
  
• 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

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;
  

2x QuantiTect SYBR Green PCR Master Mix	22.5μL
1/20xRT products	4.5μL
MilliQ	9μL
total	36μL

• 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℃ for 30 min. If the colonies of E.coli transformed with the Control
  

Restrictive Digestion

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

• Mix the following.

Sample	5µL
10xBuffer	1µL
Restriction Enzyme	0.1µL
MilliQ	-3.9µL

• Let stand for 2h at 37℃
  

Media

M9 medium

• Stir Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g and NH4Cl 1 g with water 1L.
  
• After autoclave, add 10 ml filter sterilized 100 mM MgSO4, 20 % glucose, 10 mM CaCl2, and 100 mM thiamine-HCl.
  

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

• Stir Tryptone 20 g, Yeast extract 10 g and NaCl 10 g with water 200ml.
  
• If you make LB plates, add agar 10 g.
  
• Autoclave.
  

SOB medium

• Stir Tryptone 20g and Yeast extract 5g with water.
  
• Add 2 ml 5 M NaCl and 840 ul 3 M KCl and add water up to 1L.
  
• 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.
  

Buffer TB

• Stir PIPES and CaCl2・2H2O with 100 ml water.
  
• Add 8.315 ml 3 M KCl.
  
• Add KOH and adjust pH 6.8.
  
• Add MnCl2・4H2O.
  
• Add water up to 200 ml.
  
• Filter sterilize.
  

Solubilization of Antibiotics

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

Ampicillin

Ampicillin	1.0g
MilliQ	20mL

Kanamycin

Kanamycin	0.5g
MilliQ	10mL

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

Transformation

• Unfreeze conpitent cells on ice.
  
• Dry a plate by letting the plate upside down and partly open in incubator.
  
• Add 1µL DNA solution and 20µL 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 60s at 42℃.
  
• 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.
  

Sequence

Use Big Dye Terminator 3.1（ABI）

• Mix the following
  

5xBuffer	2µL
Primer (3.2µM)	1µL
Template Plasmid	200ng
Big Dye Terminator 3.1	0.5µL
MilliQ	up to 10µL

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

Golden Gate Assembly Protocol

• 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:

linearized vector backbone	each additional assembly piece	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 Ligase, 2 million cohesive end units / mL	dH2O
100 ng	to equimolar with backbone	1.5 µl	0.15 µl	1µl	1 µl	to 15µl

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.

Material

Strains

E.coli

JM109