Team:Kyoto/Calendar/24 July 2009

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Contents

Lab-note on 24 July 2009

ExperimenterKashima, Takashima, Hibino, Miyazawa
Note-takerTakashima

Title

7/24 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 2mins at 15000g 4℃.
  3. Removed the top of layer.
  4. Centrifuged 2mins at 15000g 4℃.
  5. Removed the top of layer.
  6. Centrifuged 10mins at 14000g 4℃.
  7. Applied the top of layer to column.
  8. Centrifuged RT, 10sec and remove through.
  9. Added Buffer PB 500µl.
  10. Centrifuged RT, 10sec and remove through.
  11. Added Buffer PE 650µl.
  12. Centrifuged RT, 10sec and remove through.
  13. Centrifuged 1min at 17900g 4℃.
  14. Exchanged tube. (remove Ethanol)
  15. Added Milli Q 50µl RT 1min.
  16. Centrifuged 1min at 17900g 4℃.
  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 ℃ (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

3-O-23-A-4
Parts length / bp 2837487
Plasmid length / bp 32663266



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