Difference between revisions of "Team:KAIT Japan/Protocol"

Latest revision as of 15:24, 18 September 2015

Protocol

[SDS-PAGE]
1) Prepare the gel
2) Place stacking gel on separating gel
3) Fill electrophoresis chamber electrophoretic buffer
4) Add 4×sample buffer the cooldshock expressed protein sample and heat block (95℃,10minutes)
5) Load prepared sample into wells
6) Run the electrophoresis in 15mA, 1 hour 25 minutes
7) Dye the gel with CBB for 30 minutes
8) Branch the gel for 2 hours while shaking it

[Native-PAGE]
1) Prepare the gel
2) Place stacking gel on separating gel
3) Fill electrophoresis chamber electrophoretic buffer
4) Add 3×sample buffer
5) Load prepared sample into wells
6) Run the electrophoresis in 15mA, 1 hour 25 minutes
7) Dye the gel with CBB for 30 minutes
8) Branch the gel for 2 hours while shaking it

[Observation by confocal laser scanning microscope]
1) Irradiate the coldshock expressed protein sample with light of 400nm
2) Irradiate the coldshock expressed protein sample with light of 500nm
3) Measure the time of swiching ON / OFF

[His-tag protein purification]
1) When OD600 of bacterial culture is 0.4~0.6 , add IPTG until final concentration of IPTG is 1mM and incubate at 37°C for 3 h or at 25°C overnight until OD600 of bacterial culture is 8.0
2) Add 70μL of the FastBreak Reagent /DNase I Solution to 700μL of the bacterial culture
3) Add 75μL of resuspended HisLink Resin
4) Incubate the sample and resin for 30 min, mixing frequently on a rotating platform or shaker to optimize binding
5) Place a Spin Column onto a Collection Tube and transfer the lysate and resin to column
6) Centrifuge the column for 5 sec
7) Transfer the flow-through to a new tube
8) Add 500μL of HisLink Binding/Wash Buffer the column and Centrifuge the column for 5 sec
9) Repeat for a total of two washes
10) Place the spin column onto a new tube and Add 200μL of HisLink Elution Buffer
11) Tapping and wait 3 min
12) Centrifuge the column at 14,000rpm for 1 min

[Miniprep]
1) Culture bacterial strain with the LB medium which I added chloramphenicol to so that density becomes 100ug/mL overnight(We made a nutrient medium of around 5 mL in 50 mL falcons)(Against 5 mL of nutrient mediums, Chl used 5μL)
2) Aliquot 1mL culture into a 1.5 mL tube, and make it spin at 15000rpm (4℃) for 1 min to harvest the bacteria
3) Remove supernatant and performed 2)operation again, remove supernatant
4) Resuspend bacterial pellet by complete vortexing in 100μL of SolutionⅠ
5) Invert bacterial pellet by complete fall mixtureing in 200μL of SolutionⅡ, and confirme that it became transparent
6) Cool for 3 min on ice
7) Invert bacterial pellet by complete fall mixtureing in 150μL of SolutionⅢ, and confirme that it became cloudiness
8) Cool for 3 min on ice
9) Harvest the DNA by spinning at 15000rpm (4℃) for 10 min
10) Gather 430μL of supernatant and move it in a new tube
11) Add 0.5μL RNase(100μg/mL) and incubate the solution(37℃,1min)
12) Add 200μL phenol:chloroform(1:1) and invert
13) Harvest by spinning at 15000rpm (4℃) for 5 min
14) Remove 400μL of supernatant and move it in a new tube, after that tapped in 200μL of chloroform
15) Harvest by spinning at 15000rpm (4℃) for 1 min
16) Move 360μL of supernatant to a new tube and add 36μL of 3M CH3COONa
17) Add 925μL of 100% ethanol and made it stirred well
18) Harvest by spinning at 15000rpm (4℃) for 20 min
19) Remove only supernatant and add 400μL of 70% ethanol
20) Harvest by spinning at 15000rpm (4℃) for 20 min
21) Remove only supernatant and open the cover of the tube for 10min to dry ethanol
22) Add 20μL of TE to dissolve DNA

[PCR]

[DNA purification]
1) Place a FADF Column into a Collection Tube
2) Add FADF buffer to DNA sample which wants to purify
3) Transfer the liquid to FADF column and centrifuged for 30 sec, then discard the flow-through
4) Add 700μL of Wash buffer to FADF column and centrifuged for 30 sec, then discard the flow-through
5) Centrifuge the column for an additional 3 min to dry the column matrix
6) Place the FADF Column to a new micro tube
7) Add 40μL of D2W to the membrane center of the FADF Column
8) Stand the FADF Column for 2 min and centrifuge for 2 min to elute the DNA

[Restriction digest]
1) Add 2μL of 10×restriction enzyme buffer
2) Add 1μL each restriction enzyme
3) Add DNA sample
4) Add D2W up to 20μL
5) Heat block the reaction solution(37℃,2 h )

[Ligation]
1) Add DNA mix(insert ＋ vector)
2) Add 1μL of T4 DNA Ligase and 1/2 of total reaction solution of 2×T4 DNA Ligase buffer
3) Ligation for 15 min at room temperature

[Transformation]
1) Add plasmid to competent cell
2) Tapping and leave the competent cell on ice 30 min
3) Heat shocked 30 sec
4) Leave on ice for 2 min
5) Add 250μL of SOC medium and recovery culture(37℃, 1 h)
6) Apply 10μL of the bacteria liquid to LB agar nutrient medium after applied 20μL of chloramphenicol

[Coldshock expression]
1) Add chloramphenicol to LB medium
2) Pick up the transformant and Add it to the LB medium
3) Shake culture at 37℃ until OD600 is 0.4～0.5
4) Cool at 15℃ for 30 min
5) Add IPTG(100mM) to the cooled LB medium until final concentration of IPTG is 0.1
6) Shake culture at 15℃ for 24 h

[Sequence]
1) Perform PCR amplification o using biobrick primer(VF2 and VR). We would like to amplify the Dronpa 145N and Dronpa 145K containg psB1C3 Plasmid.

2) Purify DNA using gel extraction. Please see [DNA purification] column.
3) Perform cycle sequencing.Table.1 reaction reagent

4) Purify sequencing products.
1] Prepare the 96wellplate.
2] Add the full volume sample and Agent Clean SEQ 10 µL and 85 %ethanol 42 µL.
3] Do pipetting seventh.
4] Wait 5min.
5] Remove supernatant.
6] Add 85%ethanol 100uL to 96well plate.
7] Wait 30 sec.
8] Remove supernatant.
9] Repeat 6) ~ 8).
10] Dry 10 min at room temperature.
11] Add D₂W 40 µL to 96 well plate.
12] Incubate 10 min at room temperature.
13] Place the 96wellplate on the fragile plate.
14] Wait 5 min.
15] Transform supernatant 30µL to new 96wellplate.

5) Perform capillary electrophoresis.

---Link list---

---Sponsor---

@@ Line 263: / Line 263: @@
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+<strong>[Sequence]</strong><br>
+)      Perform PCR amplification o using biobrick primer(VF2 and VR). We would like to amplify the Dronpa 145N and Dronpa 145K containg psB1C3 Plasmid.<br>
+<br>
+<img src="https://static.igem.org/mediawiki/2015/5/55/Purotokoru2222222222222222222.PNG"><br>
+<br>
+)      Purify DNA using gel extraction. Please see [DNA purification] column.<br>
+)      Perform cycle sequencing.Table.1 reaction reagent<br>
+<br>
+<img src="https://static.igem.org/mediawiki/2015/9/97/Purotokoooooooool.PNG"><br>
+<br>
+)      Purify sequencing products.<br>
+] Prepare the 96wellplate.<br>
+] Add the full volume sample and Agent Clean SEQ 10 µL and 85 %ethanol 42 µL.<br>
+] Do pipetting seventh.<br>
+] Wait 5min.<br>
+] Remove supernatant.<br>
+] Add 85%ethanol 100uL to 96well plate.<br>
+] Wait 30 sec.<br>
+] Remove supernatant.<br>
+] Repeat 6) ~ 8).<br>
+] Dry 10 min at room temperature.<br>
+] Add D₂W 40 µL to 96 well plate.<br>
+] Incubate 10 min at room temperature.<br>
+] Place the 96wellplate on the fragile plate.<br>
+] Wait 5 min.<br>
+] Transform supernatant 30µL to new 96wellplate.<br>
+<br>
+)       Perform capillary electrophoresis.
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+<font size="9" face="Freestyle Script">---Link list---</font>
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