Team:Kyoto/Calendar/24 July 2009

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Lab-note on 24 July 2009

ExperimenterKashima, Takashima, Hibino, Miyazawa
Note-takerTakashima

Title

24 July: Miniprep Kit, Restriction Enzyme Digestion Electrophoresis

Main Protocol

  1. Miniprep Kit
  2. Restriction Enzyme Digestion
  3. Electrophoresis

Storage list

Sample DateSample NameEnzymepurification KitProductStorage
iGEM DNA parts kit3-O-2Eco R1 6F freezer
iGEM DNA parts kit3-A-4Eco R1 6F freezer
iGEM DNA parts kit3-O-2Eco R1
PST1		 6F freezer
iGEM DNA parts kit3-A-4Eco R1
PST1		 6F freezer

Miniprep

Protocol

  • [3-O-2, 3-A-4]
  1. Cultured samples (090722 9:30 - 090723 16:50)
  2. Centrifuged 2 mins at 15000 G 4 °C.
  3. Removed the top of layer.
  4. Centrifuged 2 mins at 15000 G 4 °C.
  5. Removed the top of layer.
  6. Centrifuged 10 mins at 14000 G 4 °C.
  7. Applied the top of layer to column.
  8. Centrifuged RT, 10 sec and remove through.
  9. Added Buffer PB 500 µl.
  10. Centrifuged RT, 10 sec and remove through.
  11. Added Buffer PE 650 µl.
  12. Centrifuged RT, 10 sec and remove through.
  13. Centrifuged 1min at 17900 G 4 °C.
  14. Exchanged tube. (remove Ethanol)
  15. Added Milli Q 50 µl RT 1min.
  16. Centrifuged 1min at 17900 G 4°C.
  17. Collected through and measured concentration.

Results

Sampleconc./(ng/µl)
3-O-2283.5
3-A-4103.9

Electrophoresis

Protocol

  • Gel:Agarose
  • Time:12:50-13:20
  • Voltage:100 V
1234
Sample 3-O-2 (3 µl)3-A-4 (3 µl)3-O-2 (3 µl)3-A-4 (3 µl)
enzyme Eco R1 (0.17 µl)Eco R1 (0.17 µl)Eco R1 (0.17 µl)Eco R1 (0.17 µl)
enzyme PST1 (0.3 µl)PST1 (0.3 µl)
Water / µl 1 1 1.41.4
H buffer / µl 8.28.28.18.1
Total / µl 12.712.712.912.9

Incubated at 37 °C (090724 12:50 to 13:20 )

12345
Sample 3-O-23-A-43-O-23-A-4Marker
volume / µl 101013132.5
6 times loddingbuffer / µl 222.62.60.5

Results

Result of Electrophoresis.
3-O-23-A-4
Parts length / bp 2837487
Plasmid length / bp 32663266

Discussion

  • This result indicates that the parts ( 3-O-2, 3-A-4) were treated with enzyme in appropriate way.



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