Team:KIT-Kyoto/Notebook-Protocol

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Revision as of 13:26, 7 September 2012 by Kazuko (Talk | contribs)







Midiprep by Pure LinkTM HiPure Midiprep Kit

1. 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 electrophoresis
Purification of DNA from gel by QIA quick Gel Extraction Kit

1. 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