Team:KIT-Kyoto/Notebook-Protocol
From 2012.igem.org
LB medium*per kilogram
1. Mix the reagents according to the previous components. 2. Adjust the pH (pH7.0) with NaOH. 3. Autoclave at 120℃ for 20min LB plate*per liter
1. Mix the reagent according to the previous components. 2. Add NaOH and carry the enzymes pH7.0. 3. Add 15g Bacto agar and dissolve. 4. Autoclave at 120℃ 20min. 5. After cooling down approx. 60℃, add the suitable amounts of appropriate antibiotics. 6. Pour 20~30mL into laboratory dishes. *Solution Ⅰ
*Solution Ⅱ
*Solution Ⅲ
Methods Transformation into plasmid of E.coli.1. Dissolve competent cell on ice. 2. Pour 100uL competent cell into 1.5mL tube, and mix with 1~5uL DNA. 3. Incubate for 30 minutes on ice. 4. Treat with heat shock the cells at 42℃ for 45 seconds. 5. Cool down on ice for 2 minutes. 6. Add 250uL S.O.C medium at room temperature. 7. Incubate at 37℃ with shaking for 1 hour. 8. Spread a suitable amount of the cells on LB plate (containing appropriate antibiotics). 9. Incubate overnight at 37℃. Picking up a single colonyPick up the colony grown on the LB plate by a platinum stick and put in 2.5mL of LB medium (containing appropriate antibiotics), and cultivate cells at 37˚C. Midiprep by Pure LinkTM HiPure Midiprep Kit1. Centrifuge (4000xg,4°C,10min) E. coli culture in LB medium (50 mL) and remove the supernatant 2. Mix with suspension buffer (4mL) including RNase (20 micro g/mL) and the pellet 3. Add Lysis buffer (4mL) and turn upside and down several times in order to mix well 4. Incubate at room temperature for 5min 5. Add precipitation buffer (4mL) and shake the tube quickly 6. Centrifuge and remove the supernatant (15000 x g, room temperature, 10 min) 7. Transfer the supernatant to balanced column and elute out the solution by gravity 8. Wash the column twice with Wash buffer (10 mL) and elute out the solution as gravitation every after washing 9. Put sterilized 15 mL centrifuge tube under the column 10. Add Elution buffer (5 mL) to the column and elute out the solution by gravity 11. Add isopropanol (3.5 mL) to the centrifuge tube and mix well 12. Centrifuge and remove the supernatant (15000 x g, 4°C, 30min) 13. Add 70% ethanol and mix well 14. Centrifuge and remove the supernatant (15000 x g, 4°C, 5min) 15. Dehydrated ,then suspend purified DNA into TE buffer (200 uL) Agarose gel electrophoresisPurification of DNA from gel by QIA quick Gel Extraction Kit1. Cut out the DNA fragment from agarose gel and put it in a 1.5mL tube and weigh the gel 2 .Add Buffer QG (x 3 volume of the gel weight) 3 .Vortex every 2 - 3 minutes to dissolve the gel completely 4 . Add isopropanol (equal weight to the gel, 100uL per 100mg) 5 . Trabsfer to spin column 6 . Centrifuge (10000rpm, room temperature,1min) 7 . Remove the solution 8 . Add Buffer PE (750 uL) and centrifuge (10000rpm,room temperature,1min) 9 . Remove the solution and centrifuge (17900 x g, room temperature, 1min) 10. Set a new 1.5 tube under the column, add TE (30uL) and centrifuge (10000 rpm, room temperature,1min) 11. Collect the purified DNA BP reaction by Invitrogen gateway system1. PCR using primers containing the attB sequence. 2. Purify PCR product. 3. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:
4. Vortex BP ClonaseII enzyme mix briefly. Add 1 - 2 μL to the components above and mix well by vortexing and spin them down. 5. Incubate reaction at 25˚C for more than 1 hour. 6. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly. 7. Incubate at 37˚C for 10 minutes. LR reaction by Invitrogen gateway system1. Add the following components to a 1.5 mL microcentrifuge tube at room temperature and mix:
2. Vortex LR ClonaseII enzyme mix briefly. Add 1 – 2 μL, to the components above and mix well by vortexing and spin down. 3. Incubate reaction at 25˚C for 16 hours. 4. Add 1 μL of 2 μg/μL Proteinase K solution and vortex briefly. 5. Incubate at 37˚C for 10 minutes. |