Team:Kyoto/Material and method

From 2013.igem.org

(Difference between revisions)
(Electrophoresis)
Line 14: Line 14:
==PCR==
==PCR==
'''PCR: ToYoBo KOD FX or ToYoBo KOD PLUS'''<br>
'''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 template DNA. If the concentration of DNA is 2-100ng/&micro;L, transfer 1&micro;L to a clean tube and add 99&micro;L 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>
+
*Dilute Primer. If the concentration of Primer is X&micro;M, dilute primer X timers and transfer 1&micro;L to a clean tube and add 99&micro;L MilliQ.<br>
*Mix the following.<br>
*Mix the following.<br>
'''For use of KOD plus ver2'''<br>
'''For use of KOD plus ver2'''<br>
{|class="wikitable"
{|class="wikitable"
-
|25mM MgSO4||3µL
+
|25mM MgSO4||3&micro;L
|-
|-
-
|2mM dNTPs||5µL
+
|2mM dNTPs||5&micro;L
|-
|-
-
|10xBuffer for KOD plus ver.2||5µL
+
|10xBuffer for KOD plus ver.2||5&micro;L
|-
|-
-
|Template DNA (5ng/µL)||5µL
+
|Template DNA (5ng/&micro;L)||5&micro;L
|-
|-
-
|Primer Forward (10µM)||1.5µL
+
|Primer Forward (10&micro;M)||1.5&micro;L
|-
|-
-
|Primer Reverse (10µM)||1.5µL    
+
|Primer Reverse (10&micro;M)||1.5&micro;L    
|-
|-
-
|KOD plus ver.2||1µL
+
|KOD plus ver.2||1&micro;L
|-
|-
-
|MilliQ||28µL
+
|MilliQ||28&micro;L
|-
|-
-
|Total||50µL
+
|Total||50&micro;L
|}
|}
'''For use of KOD FX'''
'''For use of KOD FX'''
{|class="wikitable"
{|class="wikitable"
-
|2mM dNTPs||10µL
+
|2mM dNTPs||10&micro;L
|-
|-
-
|2xBuffer for KOD FX||25µL    
+
|2xBuffer for KOD FX||25&micro;L    
|-
|-
-
|Template DNA||5µL
+
|Template DNA||5&micro;L
|-
|-
-
|Primer Forward (10µM)||1.5µL    
+
|Primer Forward (10&micro;M)||1.5&micro;L    
|-
|-
-
|Primer Reverse (10µM)||1.5µL    
+
|Primer Reverse (10&micro;M)||1.5&micro;L    
|-
|-
-
|KOD FX||1µL
+
|KOD FX||1&micro;L
|-
|-
-
|MilliQ||6µL    
+
|MilliQ||6&micro;L    
|-
|-
-
|Total||50µL  
+
|Total||50&micro;L  
|}
|}
-
*Let stand for 2min at 94℃.<br>
+
*Let stand for 2min at 94&deg;C.<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>
+
*25-40 cycles for 10s at 98&deg;C, for 30s at Tm-5&deg;C, and for 1min (1min for 1kb) at 68&deg;C (Tm is temperature at which primer will dissolve).<br>
*Agarose Gel Electrophoresis for confirmation.<br><br>
*Agarose Gel Electrophoresis for confirmation.<br><br>
'''PCR: Takara Ex taq'''<br>
'''PCR: Takara Ex taq'''<br>
*Mix the following (Do on PCR Bench).<br>
*Mix the following (Do on PCR Bench).<br>
{|class="wikitable"
{|class="wikitable"
-
|10x PCR buffer (TAKARA)||40µL
+
|10x PCR buffer (TAKARA)||40&micro;L
|-
|-
-
|2.5mM dNTP||8µL
+
|2.5mM dNTP||8&micro;L
|-
|-
-
|Primer-1 (10pmol/µL)||8µL
+
|Primer-1 (10pmol/&micro;L)||8&micro;L
|-
|-
-
|Primer-2 (10pmol/µL)||8µL
+
|Primer-2 (10pmol/&micro;L)||8&micro;L
|-
|-
-
|Ex Taq HS (TAKARA)||1.6µL
+
|Ex Taq HS (TAKARA)||1.6&micro;L
|-
|-
-
|MilliQ||334µL (to total 400µL)
+
|MilliQ||334&micro;L (to total 400&micro;L)
|-
|-
-
|Total||400µL
+
|Total||400&micro;L
|}
|}
-
*Dispense 25µL to 15 tubes.<br>
+
*Dispense 25&micro;L to 15 tubes.<br>
*Pick a single colony and transfer it to each tube.<br>
*Pick a single colony and transfer it to each tube.<br>
*Suspend the colony.<br>
*Suspend the colony.<br>
-
*Let stand for 10min at 90℃.<br>
+
*Let stand for 10min at 90&deg;C.<br>
-
*35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 68℃.<br>
+
*35 cycles for 30s at 94&deg;C, for 30s 55&deg;C, and for 1min at 68&deg;C.<br>
-
*Let stand for 4min at 68℃.<br>
+
*Let stand for 4min at 68&deg;C.<br>
*Add 5mL Loading Buffer to the tubes.<br>
*Add 5mL Loading Buffer to the tubes.<br>
*Agalose Gel Electrophoresis for confirmation.<br>
*Agalose Gel Electrophoresis for confirmation.<br>
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*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>
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==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; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)<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 10 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 5 mL overnight DH5a culture into 500 mL LB media in 3 L 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 30 mins<br>
*Transfer cells to 2 centrifuge bottles (250 mL), and place cells on ice for 30 mins<br>
-
*Centrifuge cells in Sorval GSA rotor at 4℃ for 10 mins at 3,000 g.<br>
+
*Centrifuge cells in Sorval GSA rotor 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>
*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 12 mL of cold TB.<br>
-
*Centrifuge cells using Sorval RT6000B rotor at 4℃ for 10 mins at 3,000 g (2500 rpm)<br>
+
*Centrifuge cells using Sorval RT6000B rotor at 4&deg;C 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 μL of DMSO. Transfer 20 μL to 1.5 mL tube<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>
-
*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>
+
*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>
==Miniprep==
==Miniprep==
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*Transfer 1.5ml of the culture to same tube and harvest as same. Remove the medium by decanting.<br>
*Transfer 1.5ml of the culture to same tube and harvest as same. Remove the medium by decanting.<br>
*Transfer 1.0ml of the culture to same tube and harvest as same. Remove the medium by pipetting.<br>
*Transfer 1.0ml of the culture to same tube and harvest as same. Remove the medium by pipetting.<br>
-
*Resuspend pelleted bacterial cells in 250µL Buffer S1 and mix thoroughly by pipeting.<br>
+
*Resuspend pelleted bacterial cells in 250&micro;L Buffer S1 and mix thoroughly by pipeting.<br>
-
*Add 250µL Buffer S2 and mix thoroughly by inverting the tube gently 4-6 times.<br>
+
*Add 250&micro;L Buffer S2 and mix thoroughly by inverting the tube gently 4-6 times.<br>
-
*Add 250µL Buffer S3 and mix immediately and thoroughly by inverting the tube 4-6 times.<br>
+
*Add 250&micro;L Buffer S3 and mix immediately and thoroughly by inverting the tube 4-6 times.<br>
*Centrifuge for 10min at 15,000g at 4°C.<br>
*Centrifuge for 10min at 15,000g at 4°C.<br>
*Apply the supernatants from step 10 to the  spin column by decanting.<br>
*Apply the supernatants from step 10 to the  spin column by decanting.<br>
Line 133: Line 133:
*Wash the spin column by adding 0.75mL Buffer PW and centrifuging for 1min at 15,000g at 4°C.<br>
*Wash the spin column by adding 0.75mL Buffer PW and centrifuging for 1min at 15,000g at 4°C.<br>
*Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.<br>
*Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.<br>
-
*Place the column in a clean tube. To elute DNA, add 30µL water to the center of each spin column, let stand for 1min, and centrifuge for 1min.<br>
+
*Place the column in a clean tube. To elute DNA, add 30&micro;L water to the center of each spin column, let stand for 1min, and centrifuge for 1min.<br>
*Discard the spin column.<br>
*Discard the spin column.<br>
*Measure the concentration of DNA by using eppendorf BioPhotometer plus or nanodrop.<br>
*Measure the concentration of DNA by using eppendorf BioPhotometer plus or nanodrop.<br>
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==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>
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*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 10x Loading Buffer for every 9µL 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>
*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>
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まだ書かれていない
まだ書かれていない
Use Wizard SV Gel and PCR Clean-Up System by Promega.<br>
Use Wizard SV Gel and PCR Clean-Up System by Promega.<br>
-
*Combine 1 part sample (PCR product) with one part Membrane Binding Solution (e.g. 50μl sample+ 50μl).<br>
+
*Combine 1 part sample (PCR product) with one part Membrane Binding Solution (e.g. 50&micro;l sample+ 50&micro;l).<br>
*Apply the solution to the column, and let stand for 1min.<br>
*Apply the solution to the column, and let stand for 1min.<br>
*Centrifuge for 1min at 13000rpm. Discard the flow-through.<br>
*Centrifuge for 1min at 13000rpm. Discard the flow-through.<br>
-
*Add 700µl Membrane Wash Solution.<br>
+
*Add 700&micro;l Membrane Wash Solution.<br>
*Centrifuge for 1min and discard the through.<br>
*Centrifuge for 1min and discard the through.<br>
-
*Add 500μl Membrane Wash Solution.<br>
+
*Add 500&micro;l Membrane Wash Solution.<br>
*Centrifuge for 5min and discard the through.<br>
*Centrifuge for 5min and discard the through.<br>
*Place the column in a clean tube.<br>
*Place the column in a clean tube.<br>
-
*Add 60µl MilliQ to the center of each column, let stand for 1min.<br>
+
*Add 60&micro;l MilliQ to the center of each column, let stand for 1min.<br>
*Centrifuge for 1min at 13000rpm.<br>
*Centrifuge for 1min at 13000rpm.<br>
*Discard the column.<br>
*Discard the column.<br>
Line 194: Line 194:
==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 215: Line 215:
==Ligation==
==Ligation==
-
*Make 2µL of Mixture (the vector and the insert at : 5-10) and Control (only the vector).<br>
+
*Make 2&micro;L of Mixture (the vector and the insert at 1 : 5-10) and Control (only the vector).<br>
-
*Add 5µL Ligation High, 1µL T4 Kinase, and 7µL MilliQ to create a solution.<br>
+
*Add 5&micro;L Ligation High, 1&micro;L T4 Kinase, and 7&micro;L MilliQ to create a solution.<br>
-
*Incubate at 16℃ for 30 min. If the colonies of ''E.coli'' transformed with the Control<br>
+
*Incubate at 16&deg;C for 30 min. If the colonies of ''E.coli'' transformed with the Control<br>
==Restriction Enzyme Digestion==
==Restriction Enzyme Digestion==
Line 223: Line 223:
*Mix the following.
*Mix the following.
{|class="wikitable"
{|class="wikitable"
-
|Sample||5µL
+
|Sample||5&micro;L
|-
|-
-
|10xBuffer||1µL
+
|10xBuffer||1&micro;L
|-
|-
-
|Restriction Enzyme||0.1µL
+
|Restriction Enzyme||0.1&micro;L
|-
|-
-
|MilliQ||-3.9µL
+
|MilliQ||-3.9&micro;L
|}
|}
-
*Let stand for 2h at 37℃<br>
+
*Let stand for 2h at 37&deg;C<br>
==Media==
==Media==
Line 238: Line 238:
*If you make M9 plates, add agar 15 g.<br>
*If you make M9 plates, add agar 15 g.<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>
*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>
+
If you need, add 10 mL filter sterilized 20 % casamino acid.<br>
'''LB medium'''<br>
'''LB medium'''<br>
*Stir BactoTryptone 2 g, Bacto-yeast extract 1 g, NaCl 1 g and 1M NaOH 200 &micro;L 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>
Line 276: Line 276:
|}
|}
*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==
Line 291: Line 291:
*Mix the following<br>
*Mix the following<br>
{|class="wikitable"
{|class="wikitable"
-
|5xBuffer||2µL
+
|5xBuffer||2&micro;L
|-
|-
-
|Primer (3.2µM)||1µL
+
|Primer (3.2&micro;M)||1&micro;L
|-
|-
|Template Plasmid||200ng
|Template Plasmid||200ng
|-
|-
-
|Big Dye Terminator 3.1||0.5µL
+
|Big Dye Terminator 3.1||0.5&micro;L
|-
|-
-
|MilliQ||up to 10µL
+
|MilliQ||up to 10&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>
+
*35 cycles for 5s at 98&deg;C, for 5s 50&deg;C, and for 2.5min 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==
==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
|}
|}
Line 323: Line 323:
5 min @ 50 C }<br>
5 min @ 50 C }<br>
5 min @ 80 C }    1 cycle<br>
5 min @ 80 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=
=Material=

Revision as of 07:22, 4 September 2013

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Material and Method Safety Attributions

Contents

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 MgSO43µL
2mM dNTPs5µL
10xBuffer for KOD plus ver.25µL
Template DNA (5ng/µL)5µL
Primer Forward (10µM)1.5µL
Primer Reverse (10µM)1.5µL
KOD plus ver.21µL
MilliQ28µL
Total50µL

For use of KOD FX

2mM dNTPs10µL
2xBuffer for KOD FX25µL
Template DNA5µL
Primer Forward (10µM)1.5µL
Primer Reverse (10µM)1.5µL
KOD FX1µL
MilliQ6µL
Total50µL
  • Let stand for 2min at 94°C.
  • 25-40 cycles for 10s at 98°C, for 30s at Tm-5°C, and for 1min (1min for 1kb) at 68°C (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 dNTP8µL
Primer-1 (10pmol/µL)8µL
Primer-2 (10pmol/µL)8µL
Ex Taq HS (TAKARA)1.6µL
MilliQ334µL (to total 400µL)
Total400µL
  • Dispense 25µL to 15 tubes.
  • Pick a single colony and transfer it to each tube.
  • Suspend the colony.
  • Let stand for 10min at 90°C.
  • 35 cycles for 30s at 94°C, for 30s 55°C, and for 1min at 68°C.
  • Let stand for 4min at 68°C.
  • 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°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 the 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 Cat No. CMP0112 by COSMO Genetech

  • 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 160rpm for 8h at 37°C with vigorous shaking.
  • Transfer 1.5ml of the culture to a tube.
  • Harvest the bacterial cells by centrifugation at 15,000g for 1min at 4°C. Remove the medium by decanting.
  • Transfer 1.5ml of the culture to same tube and harvest as same. Remove the medium by decanting.
  • Transfer 1.0ml of the culture to same tube and harvest as same. Remove the medium by pipetting.
  • Resuspend pelleted bacterial cells in 250µL Buffer S1 and mix thoroughly by pipeting.
  • Add 250µL Buffer S2 and mix thoroughly by inverting the tube gently 4-6 times.
  • Add 250µL Buffer S3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  • Centrifuge for 10min at 15,000g at 4°C.
  • Apply the supernatants from step 10 to the spin column by decanting.
  • Centrifuge for 1min at 15,000 at 4°C. Discard the flow-through.
  • Wash the spin column by adding 0.75mL Buffer PW and centrifuging for 1min at 15,000g at 4°C.
  • Discard the flow-through, and centrifuge for and additional 1min to remove residual wash buffer.
  • Place the column in a clean tube. To elute DNA, add 30µL water to the center of each spin column, let stand for 1min, and centrifuge for 1min.
  • Discard the spin column.
  • Measure the concentration of DNA by using eppendorf BioPhotometer plus or nanodrop.
  • Restriction Digestion.
  • Agarose Gel Electrophoresis for Confirmation.

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;
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

  • 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.
Sample5µL
10xBuffer1µL
Restriction Enzyme0.1µL
MilliQ-3.9µL
  • 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

Ampicillin1.0g
MilliQ20mL

Kanamycin

Kanamycin0.5g
MilliQ10mL
  • 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
5xBuffer2µL
Primer (3.2µM)1µL
Template Plasmid200ng
Big Dye Terminator 3.10.5µL
MilliQup to 10µL
  • 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:
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.

Material

Strains

E.coli(K12)

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