Team:KAIT JAPAN/protocol

PROTOCOL

Minipreparation
1) Culture bacterial strain with the LB medium which chloramphenicol is added so that density becomes 100μg/mL overnight (We made a nutrient medium around 5 mL in 50 mL falcons)(Against 5 mL of nutrient mediums, 5μL chloramphenicol is added)
2) Aliquot 1mL culture into a 1.5 mL tube, and make it spin at 14000rpm (4℃) for 1 min to harvest the bacteria
3) Remove supernatant and performed 2) operation again, remove supernatant
4) Resuspend bacterial pellet by vortexing in 100μL of Solution Ⅰ
5) Invert bacterial pellet by complete fall mixturing in 200μL of Solution Ⅱ, and make sure it became transparent
6) Cool for 3 min on ice
7) Invert bacterial pellet by complete fall mixturing in 150μL of Solution Ⅲ, and confirm that it became cloudiness
8) Cool for 3 min on ice
9) Harvest the DNA by spinning at 14000rpm (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 at 37℃ for 1min
12) Add 200μL phenol : chloroform (1:1) and invert
13) Harvest by spinning at 14000rpm (4℃) for 5 min
14) Remove 400μL of supernatant and move it into a new tube, after that tap in 200μL of chloroform
15) Harvest by spinning at 14000rpm (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 mix it well
18) Harvest by spinning at 14000rpm (4℃) for 20 min
19) Remove supernatant only and add 400μL of 70% ethanol
20) Harvest by spinning at 14000rpm (4℃) for 20 min
21) Remove supernatant only and let the cover of the tube open and warm it on incubator for 10min to dry the ethanol up
22) Add 20μL of TE to dissolve DNA

Ligation
1) Add 2μL of digested plasmid backbone (25 ng)
2) Add 3μL of Part A
3) Add 3μL of Part B
4) Add 1μL of T4 DNA Ligase and 1/2 of total reaction solution of 2×T4 DNA Ligase buffer
5) Ligate for 30 min at room temperature

PCR
1) Add 0.4μL of DNA
2) Add 2μL of primer VF2
3) Add 2μL of primer VR
4) Add 2μL 10x buffer
5) Add 1.6μL of dNTP
6) Add 0.1μL of Taq
7) Add D2W up to 20μL

DNA purification
1) Excise the agarose gel with a clean scalpel. Remove the extra agarose gel to minimize the size of the gel slice.
2) Transfer up to 300mg of the gel slice into a microcentrifuge tube.
3) Add 500μL of FADF Buffer to the sample and mix by vortexing.
4) Incubate at 55℃ for 5~10 minutes and vortex the tube every 2~3 miutes until the gel slice dissolved completely.
5) Cool down the sample mixture to room temperature. And place a FADF Column into a Collection Tube.
6) Transfer 800μL of the sample mixture to the FADF Column. Centrifuge at 11000g for 30 seconds, then discard the flow-through.
7) Add 750μL of Wash Buffer (ethanol added) to the FADF Column. Centrifuge at 11000g for 30 seconds, then discard the flow-through.
8) Centrifuge again at full speed for an additional 3 minutes to dry the column matrix.
9) Place the FADF Column to a new microcentrifuge tube.
10) Add 40μL of D2W to the membrane center of the FADF Column. Stand the FADF Column for 1 min.
11) Centrifuge at full speed for 1 min to elute the DNA.

Restriction digest
1) Add 2μL of 10× restriction enzyme buffer
2) Add 0.5μL of each restriction enzyme
3) Add 1μL of DNA sample
4) Add D2W up to 20μL
5) Heat block the reaction solution (37℃, 2h30min)

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

Agarose gel electrophoresis
1) Prepare the agarose gel (1.0% , 1.5%)
2) Load the gel (6μL : 1μL of dye, 2μL of 6x dye, and D2W)
3) Run the gel (30min)
4) Gel staining with gloves (5min)
5) Rinse the gel with water (5min)

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Contact: kaitjapan@gmail.com Twitter:@KAIT_JAPAN