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Material

Parts

<groupparts>iGEM013 Kyoto</groupparts>

Strains

E.coli(K12)

JM109
XL10-gold
BL21(DE3)pLysS
JW0588

Protocol

PCR

Mix the following.

Name	Volume
25mM MgSO4	1.5 µL
2mM dNTPs	2.5 µL
10xBuffer for KOD -Plus- ver.2	2.5 µL
Template DNA	properly
Primer Forward (10 µM)	0.75 µL
Primer Reverse (10 µM)	0.75 µL
KOD -Plus-	0.5 µL
MilliQ	up to 25 µL
Total	25 µL

Put samples into Thermal Cyclers and run the following steps.

PreDenature	Denature	Anealing	Extension	cycle
94 °C	98 °C	Tm-5 °C	68 °C	--
2 min	10 sec	30 sec	1 min/kb	30 cycles

Agarose Gel Electrophoresis for confirmation.

PCR: ToYoBo Quick Taq HS DyeMix

Mix the following.

Name	Volume
Template DNA	properly
Primer Forward (10 µM)	0.2 µL
Primer Reverse (10 µM)	0.2 µL
2x Quick Taq HS DyeMix	5 µL
MilliQ	up to 10 µ
Total	10 µL

Put samples into Thermal Cyclers and run the following steps.

PreDenature	Denature	Anealing	Extension	cycle
94 °C	94 °C	Tm-5 °C	68 °C	--
2 min	30 sec	30 sec	1 min/kb	30 cycles

Agarose Gel Electrophoresis for confirmation.

Mutation PCR

Use TaKaRa PrimeSTAR Mutagenesis BasaI Kit

Dilute the concentration of template DNA with MilliQ.
Mix the following

PrimeSTAR Max Premix (2x)	25µL
Primer Forward	10pmol
Primer Reverse	10pmol
Template DNA (1ng/µL)	1~2µL
MilliQ	up to 50µL
Total	50µL

30 cycles for 10s at 98℃, for 15s 55℃, and for 40sec at 72℃.
Agalose Gel Electrophoresis for confirmation.
Negative Control: Use nothing.
Add 2L PCR products and 20L competent cells for transformation.

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
Allow cells to grow at 37°C overnight
Place one colony in 3 mL LB media (+antibiotic selection if necessary), grow overnight at 37°C
Take 2 ml LB media and save for blank. Transfer 500&micro L overnight culture into 50 mL LB media in 500 mL flask
Allow cell to grow at 37°C (250 rpm), until OD600= 0.4 (~2-3 hours)
Place cells on ice for 30 mins
Transfer cells to a centrifuge tube (50 mL), and centrifuge cells in High Speed refrigirated centrifuge at 4°C for 10 mins at 3,000 g.
Pour off media and resuspend cells in 12 mL of cold TB.
Centrifuge cells 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.

Gel Extraction and PCR Purification

Use Promega Wizard SV Gel and PCR Clean-Up System.
Gel Extraction

Perform electrophoresis using an established protocol.
Weigh a 1.5 mlL microcentrifuge tube for each DNA fragment to be isolated and record the weight.
Visualize and photograph the DNA using a long-wavelength UV lamp and an intercalating dye such as ethidium bromide. To reduce nicking, irradiate the gel for the absolute minimum time possible. Excise the DNA fragment of interest in a minimal volume of agarose using a clean scalpel or razor blade.
Transfer the gel slice to the weighed microcentrifuge tube and record the weight. Subtract the weight of the empty tube from the total weight to obtain the weight of the gel slice.
Add Membrane Binding Solution at a ratio of 10 µL of solution per 10 mg of agarose gel slice.
Vortex the mixture and incubate at 52 °C for 10 minutes or until the gel slice is completely dissolved. Vortex the tube every few minutes to increase the rate of agarose gel melting. Centrifuge the tube briefly at room temperature to ensure the contents are at the bottom of the tube. Once the agarose gel is melted, the gel will not resolidify at room temperature.
Proceed to General.

PCR Purification

Amplify target of choice using standard amplification conditions.
Add an equal volume of Membrane Binding Solution to the PCR amplification.
Proceed to General.

General

Place one SV Minicolumn in a Collection Tube for each dissolved gel slice or PCR amplification.
Transfer the dissolved gel mixture or prepared PCR product to the SV Minicolumn assembly and incubate for 1 minute at room temperature.
Centrifuge the SV Minicolumn assembly in a microcentrifuge at 16,000 g for 1 min. Remove the SV Minicolumn from the Spin Column assembly and discard the liquid in the Collection Tube. Return the SV Minicolumn to the Collection Tube.
Wash the column by adding 700 µL of Membrane Wash Solution, previously diluted with 95% ethanol, to the SV Minicolumn. Centrifuge the SV Minicolumn assembly for 1 min at 16,000 g.
Remove the SV Minicolumn assembly from the centrifuge, being careful not to wet the bottom of the column with the flowthrough. Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
Carefully transfer the SV Minicolumn to a clean 1.5 mL microcentrifuge tube. Apply 30 mL of MilliQ directly to the center of the column without touching the membrane with with the pipette tip. Incubate at room temperature for 1 min. Centrifuge for 1 min at 16,000 g.
Discard the SV Minicolumn and store the microcentrifuge tube containing the eluted DNA at 4 °C or 20 °C

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

Use ToYoBo Ligation High Ver.2.

Mix the vector DNA and the insert DNA (the vector and the insert at 1 : 5-10).
Add a half volume of Ligation High Ver.2 to the DNA solution.
Incubate at 16 °C for 1 hour (or at 4 °C for overnight).

Restriction Enzyme Digestion

Use EcoRI, XbaI, SpeI, PstI (TaKaRa).

Mix the following.

Name	Volume
Sample DNA	2 µg
Restriction Enzyme	0.5µL
10x Buffer	3 µL
(If use XbaI,) 10x BSA	3 µL
MilliQ	up to 30 µL
total	30 µL

Let stand for 1 hour at 37 °C.

Use EcoRI, XbaI, SpeI, PstI (Promega).

Mix the following.

Name	Volume
Sample DNA	2 µg
Restriction Enzyme	0.5µL
10x Buffer	3 µL
100x BSA	3 µL
MilliQ	up to 30 µL
total	30 µL

Let stand for 1 hour 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

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°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

5xBuffer	1.75µL
Primer (3.2µM)	0.5µL
Template Plasmid	400ng
Big Dye Terminator 3.1	0.5µL
MilliQ	up to 10.5µL

Let stand for 1min at 96°C.
30 cycles for 5s at 98°C, for 5s 50°C, and for 4min 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:

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.

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.

Team:Kyoto/Material and method

From 2013.igem.org

Contents

Material

Parts

Strains

E.coli(K12)

Protocol

PCR

Mutation PCR

RNA Extraction

Making Competent Cells

Miniprep

Ethanol Precipitation

Electrophoresis

Gel Extraction and PCR Purification

qRT-PCR

Ligation

Restriction Enzyme Digestion

Media

Buffer TB

Solubilization of Antibiotics

Transformation

Sequence

Golden Gate Assembly

qRT-PCR