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

Revision as of 07:38, 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

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