Team:Kyoto/Material and method

From 2013.igem.org

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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
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{{Template:Kyoto/header}}
-
!align="center"|[[Team:Kyoto|Home]]
+
<div id="kyoto-main">
-
!align="center"|[[Team:Kyoto/Team|Team]]
+
    <div class="texts">
-
!align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Kyoto Official Team Profile]
+
    <div id="material">
-
!align="center"|[[Team:Kyoto/Project|Project]]
+
=Material=
-
!align="center"|[[Team:Kyoto/Parts|Parts Submitted to the Registry]]
+
==Parts==
-
!align="center"|[[Team:Kyoto/Modeling|Modeling]]
+
<groupparts>iGEM013 Kyoto</groupparts>
-
!align="center"|[[Team:Kyoto/Notebook|Notebook]]
+
 
-
!align="center"|[[Team:Kyoto/Material_and_method|Material and Method]]
+
==Strains==
-
!align="center"|[[Team:Kyoto/Safety|Safety]]
+
===''E.coli''(K12)===
-
!align="center"|[[Team:Kyoto/Attributions|Attributions]]
+
*'''JM109'''<br>
-
|}
+
*'''XL10-gold'''<br>
 +
*'''BL21(DE3)pLysS'''<br>
 +
*'''JW0588'''<br>
 +
 
=Protocol=
=Protocol=
-
==General==
 
==PCR==
==PCR==
-
'''PCR: ToYoBo KOD FX or ToYoBo KOD PLUS'''<br>
 
-
*Dilute template DNA. If the concentration of DNA is 2-100ng/µL, transfer 1µL to a clean tube and add 99µL MilliQ.<br>
 
-
*Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µL to a clean tube and add 99µL MilliQ.<br>
 
*Mix the following.<br>
*Mix the following.<br>
-
'''For use of KOD plus ver2'''<br>
 
{|class="wikitable"
{|class="wikitable"
-
|25mM MgSO4||3µL
+
!Name||Volume
|-
|-
-
|2mM dNTPs||5µL
+
|25mM MgSO4||1.5 &micro;L
|-
|-
-
|10xBuffer for KOD plus ver.2||5µL
+
|2mM dNTPs||2.5 &micro;L
|-
|-
-
|Template DNA (5ng/µL)||5µL
+
|10xBuffer for KOD -Plus- ver.2||2.5 &micro;L
|-
|-
-
|Primer Forward (10µM)||1.5µL
+
|Template DNA||properly
|-
|-
-
|Primer Reverse (10µM)||1.5µL  
+
|Primer Forward (10 &micro;M)||0.75 &micro;L
|-
|-
-
|KOD plus ver.2||1µL
+
|Primer Reverse (10 &micro;M)||0.75 &micro;L  
|-
|-
-
|MilliQ||28µL
+
|KOD -Plus-||0.5 &micro;L
|-
|-
-
|Total||50µL
+
|MilliQ||up to 25 &micro;L
 +
|-
 +
|Total||25 &micro;L
|}
|}
-
'''For use of KOD FX'''
+
*Put samples into Thermal Cyclers and run the following steps.
 +
{| class="wikitable"
 +
!PreDenature||Denature||Anealing||Extension||cycle
 +
|-
 +
|94 &deg;C||98 &deg;C||Tm-5 &deg;C||68 &deg;C||--
 +
|-
 +
|2 min||10 sec||30 sec||1 min/kb||30 cycles
 +
|}
 +
*Agarose Gel Electrophoresis for confirmation.<br><br>
 +
 
 +
'''PCR: ToYoBo Quick Taq HS DyeMix'''<br>
 +
*Mix the following.<br>
{|class="wikitable"
{|class="wikitable"
-
|2mM dNTPs||10µL
+
!Name||Volume
|-
|-
-
|2xBuffer for KOD FX||25µL  
+
|Template DNA||properly
|-
|-
-
|Template DNA||5µL
+
|Primer Forward (10 &micro;M)||0.2 &micro;L
|-
|-
-
|Primer Forward (10µM)||1.5µL    
+
|Primer Reverse (10 &micro;M)||0.2 &micro;L    
|-
|-
-
|Primer Reverse (10µM)||1.5µL  
+
|2x Quick Taq HS DyeMix||5 &micro;L
|-
|-
-
|KOD FX||1µL
+
|MilliQ||up to 10 &micro;
|-
|-
-
|MilliQ||6µL  
+
|Total||10 &micro;L
 +
|}
 +
*Put samples into Thermal Cyclers and run the following steps.
 +
{| class="wikitable"
 +
!PreDenature||Denature||Anealing||Extension||cycle
 +
|-
 +
|94 &deg;C||94 &deg;C||Tm-5 &deg;C||68 &deg;C||--
|-
|-
-
|Total||50µL
+
|2 min||30 sec||30 sec||1 min/kb||30 cycles
|}
|}
-
*Let stand for 2min at 94℃.<br>
+
*Agarose Gel Electrophoresis for confirmation.<br>
-
*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).<br>
+
 
-
*Agarose Gel Electrophoresis for confirmation.<br><br>
+
==Mutation PCR==
-
'''PCR: Takara Ex taq'''<br>
+
Use TaKaRa PrimeSTAR Mutagenesis BasaI Kit<br>
-
*Mix the following (Do on PCR Bench).<br>
+
*Dilute the concentration of template DNA with MilliQ.<br>
 +
*Mix the following<br>
{|class="wikitable"
{|class="wikitable"
-
|10x PCR buffer (TAKARA)||40µL
+
|PrimeSTAR Max Premix (2x)||25µL
|-
|-
-
|2.5mM dNTP||8µL
+
|Primer Forward ||10pmol
|-
|-
-
|Primer-1 (10pmol/µL)||8µL
+
|Primer Reverse||10pmol
|-
|-
-
|Primer-2 (10pmol/µL)||8µL
+
|Template DNA (1ng/µL)||1~2µL
|-
|-
-
|Ex Taq HS (TAKARA)||1.6µL
+
|MilliQ||up to 50µL
|-
|-
-
|MilliQ||334µL (to total 400µL)
+
|Total||50µL
-
|-
+
-
|Total||400µL
+
|}
|}
-
*Dispense 25µL to 15 tubes.<br>
+
 
-
*Pick a single colony and transfer it to each tube.<br>
+
*30 cycles for 10s at 98℃, for 15s 55℃, and for 40sec at 72℃.<br>
-
*Suspend the colony.<br>
+
-
*Let stand for 10min at 90℃.<br>
+
-
*35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.<br>
+
-
*Let stand for 4min at 72℃.<br>
+
-
*Add 5mL Loading Buffer to the tubes.<br>
+
*Agalose Gel Electrophoresis for confirmation.<br>
*Agalose Gel Electrophoresis for confirmation.<br>
*Negative Control: Use nothing.<br>
*Negative Control: Use nothing.<br>
-
*Positive Control: Use a colony that will yield a product with these primers.<br>
+
*Add 2L PCR products and 20L competent cells for transformation.<br>
 +
 
 +
 
==RNA Extraction==
==RNA Extraction==
Line 91: Line 104:
*Add 1mL ISOGEN-LS to sample and vortex.<br>
*Add 1mL ISOGEN-LS to sample and vortex.<br>
*Store for 5min on ice.<br>
*Store for 5min on ice.<br>
-
*Add 250µL chloroform and shake vigorously for 15 sec.<br>
+
*Add 250&micro;L chloroform and shake vigorously for 15 sec.<br>
*Store for 3min. on ice.<br>
*Store for 3min. on ice.<br>
-
*Centrifuge 17400xg for 10min. at 4℃.<br>
+
*Centrifuge 17400xg for 10min. at 4&deg;C.<br>
*Transfer aqueous phase to another tube and add 0.8 volume isopropanol.<br>
*Transfer aqueous phase to another tube and add 0.8 volume isopropanol.<br>
*Store for 10min. on ice.<br>
*Store for 10min. on ice.<br>
-
*Centrifuge 17400xg for 10min. at 4℃.<br>
+
*Centrifuge 17400xg for 10min. at 4&deg;C.<br>
*Discard the supernatant.<br>
*Discard the supernatant.<br>
-
*Add 800µL 80% ethanol and vortex.<br>
+
*Add 800&micro;L 80% ethanol and vortex.<br>
-
*Centrifuge 7500xg for 5min. at 4℃.<br>
+
*Centrifuge 7500xg for 5min. at 4&deg;C.<br>
*Discard the supernatant.<br>
*Discard the supernatant.<br>
*Dry briefly.<br>
*Dry briefly.<br>
*Dissolve in nuclease-free water.<br>
*Dissolve in nuclease-free water.<br>
-
==Making Competent cells==
+
==Making Competent Cells==
-
*Streak ''E.coli'' cells on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)<br>
+
*Streak ''E.coli'' cells on an LB plate<br>
-
*Allow cells to grow at 37℃ overnight<br>
+
*Allow cells to grow at 37&deg;C overnight<br>
-
*Place one colony in 10 mL LB media (+antibiotic selection if necessary), grow overnight at 37℃<br>
+
*Place one colony in 3 mL LB media (+antibiotic selection if necessary), grow overnight at 37&deg;C<br>
-
*Take 2 ml LB media and save for blank. Transfer 5 mL overnight DH5a culture into 500 mL LB media in 3 L flask<br>
+
*Take 2 ml LB media and save for blank. Transfer 500&micro L overnight culture into 50 mL LB media in 500 mL flask<br>
-
*Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours)<br>
+
*Allow cell to grow at 37&deg;C (250 rpm), until OD600= 0.4 (~2-3 hours)<br>
-
*Transfer cells to 2 centrifuge bottles (250 mL), and place cells on ice for 20 mins<br>
+
*Place cells on ice for 30 mins<br>
-
*Centrifuge cells in Sorval GSA rotor at 4℃ for 10 mins at 3,000 g.<br>
+
*Transfer cells to a centrifuge tube (50 mL), and centrifuge cells in High Speed refrigirated centrifuge at 4&deg;C for 10 mins at 3,000 g.<br>
-
*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<br>
+
*Pour off media and resuspend cells in 12 mL of cold TB.<br>
-
*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<br>
+
*Centrifuge cells at 4&deg;C for 10 mins at 3,000 g (2500 rpm)<br>
-
*Centrifuge cells using Sorval RT6000B rotor at 4℃ for 10 mins at 3,000 g (2500 rpm)<br>
+
*Pour supernatant and resuspend cells (by pipetting) in 5 mL of cold TB and 350 &micro;L of DMSO. Transfer 20 &micro;L to 1.5 mL tube<br>
-
*Pour supernatant and resuspend cells (by pipetting) in 8 mL cold 0.1M CaCl2 containing 15% glycerol. Transfer 140 mL into (1.5 mL)<br>
+
*Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80&deg;C can be used for transformation for up to ~6 months<br>
-
*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<br>
+
==Miniprep==
==Miniprep==
-
Use QIAprep Spin Miniprep Kit Cat. No. 27104 by QIAGEN<br>
+
Use LaboPass Plasmid Mini Purification Kit.<br>
-
*Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 3mL LB medium containing the appropriate selective antibiotic.<br>
+
*Pick a single colony from a freshly streaked selective plate and inoculate a culture of about 4mL Plusgrow medium containing the appropriate selective antibiotic.<br>
-
*Incubate at 170rpm for 8h at 37℃ with vigorous shaking.<br>
+
*Incubate at 160 rpm for 8 h at 37 &deg;C with vigorous shaking.<br>
-
*Transfer a half of the culture to a tube.<br>
+
*Pellet the bacterial culture by centrifugation for 1 min at 15,000 g in a tabletop centrifuge.<br>
-
*Harvest the bacterial cells by centrifugation at 14,000g for 1min at 4℃. Remove the medium by decanting.<br>
+
*Discard the supernatant as much as possible.<br>
-
*Transfer the half of the culture to same tube and harvest as same. Remove the medium by pipetting.<br>
+
*Resuspend pelleted bacterial cells in 250 &micro;L of Buffer S1.<br>
-
*Resuspend pelleted bacterial cells in 250µL Buffer P1 and mix thoroughly by pipeting.<br>
+
*Transfer the suspension to a new 1.5 mL tube.<br>
-
*Add 250µL Buffer P2 and mix thoroughly by inverting the tube gently 4-6 times.<br>
+
*Add 250 &micro;L of Buffer S2 and mix by inverting the tube 4 times (do not vortex).<br>
-
*Add 350µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.<br>
+
*Add 350 &micro;L of Buffer S3 and immediately mix by inverting the tube 4-6 times (do not vortex).<br>
-
*Centrifuge for 10min at 14,000g at 4℃.<br>
+
*Centrifuge for 10min.<br>
-
*Apply the supernatants from step 10 to the QIAprep spin column by pipetting.<br>
+
*Transfer carefully the supernatant to a spin column and centrufuge for 1 min.<br>
-
*Centrifuge for 10s in a table-top microcentrifuge. Discard the flow-through.<br>
+
*Remove the spin column, discard the flowthrough, and re-insert the spin column to the collection tube.<br>
-
*Wash the QIAprep spin column by adding 0.5mL Buffer PB and centrifuging for 10s in a table-top microcentrifuge. Discard the flow-through.<br>
+
*Apply the 750 &micro;L of Buffer PW and centrifuge for 1 min.<br>
-
*Wash QIAprep spin column by adding 0.65mL Buffer PeE and centrifuging for 10s in a table-top microcentrifuge.<br>
+
*Remove the spin column, discard the flowthrough, and re-insert the spin column to the collection tube.<br>
-
*Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.<br>
+
*Discard the flowthrough, and centrifuge for an additional 1 min to remove residual wash buffer.<br>
-
*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.<br>
+
*Transfer the spin column to a new 1.5 mL tube.<br>
-
*Discard the QIAprep spin column.<br>
+
*Add 30 &micro;L of MilliQ, let stand for 1 min, and centrifuge for 1 min.<br>
-
*Measure the concentration of DNA by using eppendorf BioPhotometer plus.<br>
+
-
*Restriction Digestion.<br>
+
-
*Agarose Gel Electrophoresis for Confirmation.<br>
+
-
 
+
-
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.<br>
+
-
*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).<br>
+
-
*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).<br>
+
-
*Add 10µl of Alkaline Protease Solution and mix by inverting the tube 4 times. Incubate for 5 minutes at room temperature.<br>
+
-
*Add 350µl of Neutralization Solution and immediately mix by inverting the tube 4 times (do not vortex).<br>
+
-
*Centrifuge the bacterial lysate at maximum speed (around 14,000 × g) in a microcentrifuge for 10 minutes at room temperature.<br>
+
-
*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.<br>
+
-
*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.<br>
+
-
*Add 750µl of Column Wash Solution, previously diluted with 95% ethanol, to the Spin Column.<br>
+
-
*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.<br>
+
-
*Repeat the wash procedure using 250µl of Column Wash Solution.<br>
+
-
*Centrifuge at maximum speed in a microcentrifuge for 2 minutes at room temperature.om temperature.<br>
+
-
*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.<br>
+
-
*Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube.<br>
+
-
*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.<br>
+
-
*After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube and discard the Spin Column.<br>
+
-
*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.<br>
+
-
*Cap the microcentrifuge tube and store the purified plasmid DNA at –20°C or below.<br>
+
==Ethanol Precipitation==
==Ethanol Precipitation==
Use Ethachinmate (NIPPON GENE、312-01791).<br>
Use Ethachinmate (NIPPON GENE、312-01791).<br>
-
*Add 3.3 µL of 3M Sodium Acetate (attached with Ethachinmate) into 100µL of DNA solution.<br>
+
*Add 3.3 &micro;L of 3M Sodium Acetate (attached with Ethachinmate) into 100&micro;L of DNA solution.<br>
-
*Add 1µL of Ethachinmate.<br>
+
*Add 1&micro;L of Ethachinmate.<br>
*Vortex.<br>
*Vortex.<br>
-
*Add ethanol, 200-250µL.<br>
+
*Add ethanol, 200-250&micro;L.<br>
*Vortex.<br>
*Vortex.<br>
*Centrifuge at 12000xg for 5min.<br>
*Centrifuge at 12000xg for 5min.<br>
Line 187: Line 176:
*Place the tray in electrophoresis chamber.<br>
*Place the tray in electrophoresis chamber.<br>
*Cover the tray with 1xTAE buffer.<br>
*Cover the tray with 1xTAE buffer.<br>
-
*To prepare samples for electrophoresis, add 1µL of 6x Loading Buffer for every 5µL of DNA solution and mix well.<br>
+
*To prepare samples for electrophoresis, add 1&micro;L of 10x Loading Buffer for every 9&micro;L of DNA solution and mix well.<br>
-
*Load 6µL of the DNA solution per well.<br>
+
*Load 6&micro;L of the DNA solution per well.<br>
*Electrophoresis at 100V for about 30min until Loading Buffer have migrated approximately three-quarters of the gel.<br>
*Electrophoresis at 100V for about 30min until Loading Buffer have migrated approximately three-quarters of the gel.<br>
-
*Stain the gel in 0.5µg/mL ethidium bromide for 20-30min.<br>
+
*Stain the gel in 0.5&micro;g/mL ethidium bromide for 20-30min.<br>
-
*Rinse the gel with MilliQ.<br>
+
*Place a plastic wrap on the transilluminator in the cabinet of Printgraph.<br>
*Place a plastic wrap on the transilluminator in the cabinet of Printgraph.<br>
*Place the gel on the transilluminator.<br>
*Place the gel on the transilluminator.<br>
Line 199: Line 187:
*Dispose of the gel.<br>
*Dispose of the gel.<br>
-
==PCR Purification==
+
==Gel Extraction and PCR Purification==
-
Use Wizard SV Gel and PCR Clean-Up System by Promega.<br>
+
Use Promega Wizard SV Gel and PCR Clean-Up System.<br>
-
*Combine 1 part sample (PCR product) with one part Membrane Binding Solution (e.g. 50μl sample+ 50μl).<br>
+
'''Gel Extraction'''
-
*Apply the solution to the column, and let stand for 1min.<br>
+
*Perform electrophoresis using an established protocol.<br>
-
*Centrifuge for 1min at 13000rpm. Discard the flow-through.<br>
+
*Weigh a 1.5 mlL microcentrifuge tube for each DNA fragment to be isolated and record the weight.<br>
-
*Add 700µl Membrane Wash Solution.<br>
+
*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.<br>
-
*Centrifuge for 1min and discard the through.<br>
+
*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.<br>
-
*Add 500μl Membrane Wash Solution.<br>
+
*Add Membrane Binding Solution at a ratio of 10 &micro;L of solution per 10 mg of agarose gel slice.<br>
-
*Centrifuge for 5min and discard the through.<br>
+
*Vortex the mixture and incubate at 52 &deg;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.<br>
-
*Place the column in a clean tube.<br>
+
*Proceed to General.<br>
-
*Add 60µl MilliQ to the center of each column, let stand for 1min.<br>
+
 
-
*Centrifuge for 1min at 13000rpm.<br>
+
'''PCR Purification'''
-
*Discard the column.<br>
+
*Amplify target of choice using standard amplification conditions.<br>
 +
*Add an equal volume of Membrane Binding Solution to the PCR amplification.<br>
 +
*Proceed to General.<br>
 +
 
 +
'''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.<br>
 +
*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.<br>
 +
*Wash the column by adding 700 &micro;L 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.<br>
 +
*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.<br>
 +
*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.<br>
 +
*Discard the SV Minicolumn and store the microcentrifuge tube containing the eluted DNA at 4 &deg;C or 20 &deg;C<br>
==qRT-PCR==
==qRT-PCR==
Use QuantiTect SYBR green PCR kit Cat. No. 204143 by QIAGEN<br>
Use QuantiTect SYBR green PCR kit Cat. No. 204143 by QIAGEN<br>
-
*Dilute primer to 1.5μM.<br>
+
*Dilute primer to 1.5&micro;M.<br>
*Dilute RT products.<br>
*Dilute RT products.<br>
*Mix the following;<br>
*Mix the following;<br>
{|class="wikitable"
{|class="wikitable"
-
|2x QuantiTect SYBR Green PCR Master Mix||22.5μL
+
|2x QuantiTect SYBR Green PCR Master Mix||22.5&micro;L
|-
|-
-
|1/20xRT products||4.5μL
+
|1/20xRT products||4.5&micro;L
|-
|-
-
|MilliQ||9μL
+
|MilliQ||9&micro;L
|-
|-
-
|total||36μL
+
|total||36&micro;L
|}
|}
-
*Mix the reaction mix thoroughly, and dispense 36μL into 96 wells plate.<br>
+
*Mix the reaction mix thoroughly, and dispense 36&micro;L into 96 wells plate.<br>
-
*Add primer set 9μL.<br>
+
*Add primer set 9&micro;L.<br>
*Mix by inverting and voltex.<br>
*Mix by inverting and voltex.<br>
-
*Dispense 10μL into 384 wells plate and centrifuge.<br>
+
*Dispense 10&micro;L into 384 wells plate and centrifuge.<br>
*Let stand for 2min at 50°C and for 15min for 95°C.<br>
*Let stand for 2min at 50°C and for 15min for 95°C.<br>
*40 cycles for 15sec at 95°C, for 30sec at 60°C, and for 1min at 72°C.<br>
*40 cycles for 15sec at 95°C, for 30sec at 60°C, and for 1min at 72°C.<br>
Line 236: Line 235:
==Ligation==
==Ligation==
-
*Make 2µL of Mixture (the vector and the insert at : 5-10) and Control (only the vector).<br>
+
Use ToYoBo Ligation High Ver.2.
-
*Add 5µL Ligation High, 1µL T4 Kinase, and 7µL MilliQ to create a solution.<br>
+
*Mix the vector DNA and the insert DNA (the vector and the insert at 1 : 5-10).<br>
-
*Incubate at 16℃ for 30 min. If the colonies of ''E.coli'' transformed with the Control<br>
+
*Add a half volume of Ligation High Ver.2 to the DNA solution.<br>
 +
*Incubate at 16 &deg;C for 1 hour (or at 4 &deg;C for overnight).<br>
-
==Restrictive Digestion==
+
==Restriction Enzyme Digestion==
-
Use EcoRI, XbaI, SpeI, PstI, (NEB)<br>
+
'''Use EcoRI, XbaI, SpeI, PstI (TaKaRa).'''
*Mix the following.
*Mix the following.
{|class="wikitable"
{|class="wikitable"
-
|Sample||5µL
+
!Name||Volume
|-
|-
-
|10xBuffer||1µL
+
|Sample DNA||2 &micro;g
|-
|-
-
|Restriction Enzyme||0.1µL
+
|Restriction Enzyme||0.5&micro;L
|-
|-
-
|MilliQ||-3.9µL
+
|10x Buffer||3 &micro;L
 +
|-
 +
|(If use XbaI,) 10x BSA||3 &micro;L
 +
|-
 +
|MilliQ||up to 30 &micro;L
 +
|-
 +
|total||30 &micro;L
|}
|}
-
*Let stand for 2h at 37℃<br>
+
*Let stand for 1 hour at 37 &deg;C.<br>
 +
'''Use EcoRI, XbaI, SpeI, PstI (Promega).'''
 +
*Mix the following.
 +
{|class="wikitable"
 +
!Name||Volume
 +
|-
 +
|Sample DNA||2 &micro;g
 +
|-
 +
|Restriction Enzyme||0.5&micro;L
 +
|-
 +
|10x Buffer||3 &micro;L
 +
|-
 +
|100x BSA||3 &micro;L
 +
|-
 +
|MilliQ||up to 30 &micro;L
 +
|-
 +
|total||30 &micro;L
 +
|}
 +
*Let stand for 1 hour at 37 &deg;C.<br>
 +
 
==Media==
==Media==
'''M9 medium'''<br>
'''M9 medium'''<br>
-
*Stir Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g and NH4Cl 1 g with water 1L.<br>
+
*Stir Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g and NH4Cl 1 g with water 500 mL.<br>
-
*After autoclave, add 10 ml filter sterilized 100 mM MgSO4, 20 % glucose, 10 mM CaCl2, and 100 mM thiamine-HCl.<br>
+
*If you make M9 plates, add agar 15 g.<br>
-
If you need, add 10 ml filter sterilized 20 % casamino acid.<br>
+
*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.<br>
 +
If you need, add 10 mL filter sterilized 20 % casamino acid.<br>
'''LB medium'''<br>
'''LB medium'''<br>
-
*Stir Tryptone 20 g, Yeast extract 10 g and NaCl 10 g with water 200ml.<br>
+
*Stir BactoTryptone 2 g, Bacto-yeast extract 1 g, NaCl 1 g and 1M NaOH 200 &micro;L with water 200mL.<br>
-
*If you make LB plates, add agar 10 g.<br>
+
*If you make LB plates, add agar 2 g.<br>
*Autoclave.<br>
*Autoclave.<br>
'''SOB medium'''<br>
'''SOB medium'''<br>
-
*Stir Tryptone 20g and Yeast extract 5g with water.<br>
+
*Stir BactoTryptone 20 g and Bacto-yeast extract 5 g with water.<br>
-
*Add 2 ml 5 M NaCl and 840 ul 3 M KCl and add water up to 1L.<br>
+
*Add 2 mL 5 M NaCl and 840 &micro;L 3 M KCl and add water up to 1 L.<br>
-
*After autoclave, add 10 ml filter sterilized 1 M MgSO4 and 1 M MgCl2.<br>
+
*After autoclave, add 10 mL filter sterilized 1 M MgSO4 and 1 M MgCl2.<br>
'''SOC medium'''<br>
'''SOC medium'''<br>
-
*Add 2 M glucose 1 ml to 100 ml SOB.<br>
+
*Add 2 M glucose 1 mL to 100 mL SOB.<br>
 +
'''Plusgrow Medium'''<br>
 +
*Stir plusgrow 20 g and with water 500 mL.<br>
 +
*Autoclave.<br>
==Buffer TB==
==Buffer TB==
-
*Stir PIPES and CaCl2・2H2O with 100 ml water.<br>
+
*Stir 0.6g PIPES and 30mg CaCl2 with 10 mL water.<br>
-
*Add 8.315 ml 3 M KCl.<br>
+
*Add 2.5mL 2M KCl.<br>
-
*Add KOH and adjust pH 6.8.<br>
+
*Add KOH and adjust pH 6.7.<br>
-
*Add MnCl2・4H2O.<br>
+
*Add 0.218g MnCl2・4H2O.<br>
-
*Add water up to 200 ml.<br>
+
*Add water up to 20 mL.<br>
*Filter sterilize.<br>
*Filter sterilize.<br>
Line 290: Line 319:
|-
|-
|MilliQ||10mL
|MilliQ||10mL
 +
|}
 +
'''Chloramphenicol'''
 +
{|class="wikitable"
 +
|Chloramphenicol||0.5g
 +
|-
 +
|99.5% EtOH||10mL
|}
|}
*Dispense 1.1mL of the solution into 1.5mL tubes.<br>
*Dispense 1.1mL of the solution into 1.5mL tubes.<br>
-
*Store in the -20℃ freezer.<br>
+
*Store in the -20&deg;C freezer.<br>
==Transformation==
==Transformation==
-
*Unfreeze conpitent cells on ice.<br>
+
*Unfreeze conpetent cells on ice.<br>
*Dry a plate by letting the plate upside down and partly open in incubator.<br>
*Dry a plate by letting the plate upside down and partly open in incubator.<br>
-
*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.<br>
+
*Add 1~20&micro;L DNA solution and 10~50&micro;L 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.<br>
-
*Heatshock for 60s at 42℃.<br>
+
*Heatshock for 45s at 42&deg;C.<br>
*Let stand for 2min on ice.<br>
*Let stand for 2min on ice.<br>
*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.<br>
*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.<br>
 +
*Culture overnight at 37&deg;C.<br>
==Sequence==
==Sequence==
Line 306: Line 342:
*Mix the following<br>
*Mix the following<br>
{|class="wikitable"
{|class="wikitable"
-
|5xBuffer||2µL
+
|5xBuffer||1.75&micro;L
|-
|-
-
|Primer (3.2µM)||1µL
+
|Primer (3.2&micro;M)||0.5&micro;L
|-
|-
-
|Template Plasmid||200ng
+
|Template Plasmid||400ng
|-
|-
-
|Big Dye Terminator 3.1||0.5µL
+
|Big Dye Terminator 3.1||0.5&micro;L
|-
|-
-
|MilliQ||up to 10µL
+
|MilliQ||up to 10.5&micro;L
|}
|}
-
*Let stand for 1min at 96℃.<br>
+
*Let stand for 1min at 96&deg;C.<br>
-
*35 cycles for 5s at 98℃, for 5s 50℃, and for 2.5min at 68℃.<br>
+
*30 cycles for 5s at 98&deg;C, for 5s 50&deg;C, and for 4min at 68&deg;C.<br>
-
*Add 25µL 100% ethanol and 1µL NAOAC<br>
+
*Add 25&micro;L 100% ethanol and 1&micro;L NAOAC<br>
-
===Golden Gate Assembly Protocol===
+
==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:
+
*Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces to a 15 &micro;l total volume assembly reaction mixture as follows:
{|class="wikitable"
{|class="wikitable"
!linearized vector backbone||each additional assembly piece||10x NEB T4 Buffer||100x BSA||BsaI||NEB T4 Ligase, 2 million cohesive end units / mL||dH2O
!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
+
|100 ng||to equimolar with backbone||1.5 &micro;l||0.15 &micro;l||1&micro;l||1 &micro;l||to 15&micro;l
|}
|}
:NOTE: It is essential to use a High Concentration Ligase
:NOTE: It is essential to use a High Concentration Ligase
-
:BsaI is only 10% active at 37 C without the addition of BSA.
+
:BsaI is only 10% active at 37 &deg;C without the addition of BSA.
*Perform the assembly reaction in a thermocycler as follows:
*Perform the assembly reaction in a thermocycler as follows:
either (following Engler 2009):<br>
either (following Engler 2009):<br>
-
3 min @ 37 C }<br>
+
3 min @ 37 &deg;C }<br>
-
4 min @ 16 C } 25 cycles<br>
+
4 min @ 16 &deg;C } 25 cycles<br>
-
5 min @ 50 C }<br>
+
5 min @ 50 &deg;C }<br>
-
5 min @ 80 C }    1 cycle<br>
+
5 min @ 80 &deg;C }    1 cycle<br>
-
*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.
+
*Transform 5 &micro;l of the assembly reaction into 100 &micro;l of competent ''E. coli'' and/or run a diagnostic agarose gel to check for successful assembly.
-
=Material=
+
==qRT-PCR==
-
==Strains==
+
Use QuantiTect SYBR green PCR kit Cat. No. 204143 by QIAGEN<br>
-
===''E.coli''===
+
*Dilute primer to 1.5μM.<br>
-
'''JM109'''
+
*Dilute RT products.<br>
 +
*Mix the following;<br>
 +
{|class="wikitable"
 +
|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.<br>
 +
*Add primer set 9μL.<br>
 +
*Mix by inverting and voltex.<br>
 +
*Dispense 10μL into 384 wells plate and centrifuge.<br>
 +
*Let stand for 2min at 50°C and for 15min for 95°C.<br>
 +
*40 cycles for 15sec at 95°C, for 30sec at 60°C, and for 1min at 72°C.<br>
 +
*Let stand for 15sec at 95°C.<br>
 +
    </div>
 +
    </div>
 +
</div>
 +
{{Template:Kyoto/footer}}

Latest revision as of 12:47, 10 October 2013

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Contents

Material

Parts

<groupparts>iGEM013 Kyoto</groupparts>

Strains

E.coli(K12)

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

Protocol

PCR

  • Mix the following.
NameVolume
25mM MgSO41.5 µL
2mM dNTPs2.5 µL
10xBuffer for KOD -Plus- ver.22.5 µL
Template DNAproperly
Primer Forward (10 µM)0.75 µL
Primer Reverse (10 µM)0.75 µL
KOD -Plus-0.5 µL
MilliQup to 25 µL
Total25 µL
  • Put samples into Thermal Cyclers and run the following steps.
PreDenatureDenatureAnealingExtensioncycle
94 °C98 °CTm-5 °C68 °C--
2 min10 sec30 sec1 min/kb30 cycles
  • Agarose Gel Electrophoresis for confirmation.

PCR: ToYoBo Quick Taq HS DyeMix

  • Mix the following.
NameVolume
Template DNAproperly
Primer Forward (10 µM)0.2 µL
Primer Reverse (10 µM)0.2 µL
2x Quick Taq HS DyeMix5 µL
MilliQup to 10 µ
Total10 µL
  • Put samples into Thermal Cyclers and run the following steps.
PreDenatureDenatureAnealingExtensioncycle
94 °C94 °CTm-5 °C68 °C--
2 min30 sec30 sec1 min/kb30 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 Reverse10pmol
Template DNA (1ng/µL)1~2µL
MilliQup to 50µL
Total50µL
  • 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 Mix22.5µL
1/20xRT products4.5µL
MilliQ9µL
total36µL
  • 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.
NameVolume
Sample DNA2 µg
Restriction Enzyme0.5µL
10x Buffer3 µL
(If use XbaI,) 10x BSA3 µL
MilliQup to 30 µL
total30 µL
  • Let stand for 1 hour at 37 °C.

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

  • Mix the following.
NameVolume
Sample DNA2 µg
Restriction Enzyme0.5µL
10x Buffer3 µL
100x BSA3 µL
MilliQup to 30 µL
total30 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

Ampicillin1.0g
MilliQ20mL

Kanamycin

Kanamycin0.5g
MilliQ10mL

Chloramphenicol

Chloramphenicol0.5g
99.5% EtOH10mL
  • 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
5xBuffer1.75µL
Primer (3.2µM)0.5µL
Template Plasmid400ng
Big Dye Terminator 3.10.5µL
MilliQup 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 backboneeach additional assembly piece10x NEB T4 Buffer100x BSABsaINEB T4 Ligase, 2 million cohesive end units / mLdH2O
100 ngto equimolar with backbone1.5 µl0.15 µl1µl1 µlto 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 Mix22.5μL
1/20xRT products4.5μL
MilliQ9μL
total36μL
  • 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.