Team:HokkaidoU Japan/Notebook/September24

From 2010.igem.org

(Difference between revisions)
(miniprep後のサンプル泳動)
Line 7: Line 7:
#Transfered E.coli solution to 1.5 mL tubes, 1 mL each
#Transfered E.coli solution to 1.5 mL tubes, 1 mL each
-
#Centrifuged at 4C, 15000rpm for 1min
+
#Centrifuged at 4C, 15000 rpm for 1 min
#Discarded the supernatant  
#Discarded the supernatant  
#Suspended on 125 uL of Buffer P1 each
#Suspended on 125 uL of Buffer P1 each
#Added 175 uL Buffer N3 each mixed by inversion
#Added 175 uL Buffer N3 each mixed by inversion
-
#Centrifuged at 4C, 13000rpm for 10min
+
#Centrifuged at 4C, 13000 rpm for 10 min
#Transfered the supernatant to filtration column
#Transfered the supernatant to filtration column
-
#Centrifuged at 4C, 13000rpm for 1min
+
#Centrifuged at 4C, 13000 rpm for 1 min
#Discarded the flow-through
#Discarded the flow-through
#added 500 uL of Buffer PB to filtration column
#added 500 uL of Buffer PB to filtration column
-
#Centrifuged at 4C, 13000rpm for 1min
+
#Centrifuged at 4C, 13000 rpm for 1 min
#Discarded the flow-through centrifuged for 1min to remove remaining buffer
#Discarded the flow-through centrifuged for 1min to remove remaining buffer
#Transfered filtration column to a new 1.5 ml tube
#Transfered filtration column to a new 1.5 ml tube
#Resuspended on 50 ul of TE and incubated at RT for 1min
#Resuspended on 50 ul of TE and incubated at RT for 1min
-
#Centrifuged at 4C, 13000rpm for 1min
+
#Centrifuged at 4C, 13000 rpm for 1 min
== Electrophoresis of minipreped samples ==
== Electrophoresis of minipreped samples ==
#Mixed 1 uL of sample with loading buffer 1 uL
#Mixed 1 uL of sample with loading buffer 1 uL
-
#Added 6 uLof Lambda/Hind Ⅲ EcoR Marker
+
#Added 6 uL of λ/Hind Ⅲ EcoR Marker
#Electrophoresed
#Electrophoresed
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Compared to marker plasmid is about 7000 bp long
Compared to marker plasmid is about 7000 bp long
-
Arabinose Promoter+RBS+pSB1A3 is 3455bp long, so it might be tandem
+
Arabinose Promoter+RBS+pSB1A3 is 3455 bp long, so it might be tandem
From the picture estimate of concentration was about 30 ng/uL
From the picture estimate of concentration was about 30 ng/uL

Revision as of 08:37, 27 October 2010

  • Miniprep of Arac+RBS+pSB1A3
  • Follow quality check

Miniprep of Arac+RBS+pSB1A3

  1. Transfered E.coli solution to 1.5 mL tubes, 1 mL each
  2. Centrifuged at 4C, 15000 rpm for 1 min
  3. Discarded the supernatant
  4. Suspended on 125 uL of Buffer P1 each
  5. Added 175 uL Buffer N3 each mixed by inversion
  6. Centrifuged at 4C, 13000 rpm for 10 min
  7. Transfered the supernatant to filtration column
  8. Centrifuged at 4C, 13000 rpm for 1 min
  9. Discarded the flow-through
  10. added 500 uL of Buffer PB to filtration column
  11. Centrifuged at 4C, 13000 rpm for 1 min
  12. Discarded the flow-through centrifuged for 1min to remove remaining buffer
  13. Transfered filtration column to a new 1.5 ml tube
  14. Resuspended on 50 ul of TE and incubated at RT for 1min
  15. Centrifuged at 4C, 13000 rpm for 1 min


Electrophoresis of minipreped samples

  1. Mixed 1 uL of sample with loading buffer 1 uL
  2. Added 6 uL of λ/Hind Ⅲ EcoR Marker
  3. Electrophoresed


Electrophoresis of minipreped samples

Compared to marker plasmid is about 7000 bp long

Arabinose Promoter+RBS+pSB1A3 is 3455 bp long, so it might be tandem

From the picture estimate of concentration was about 30 ng/uL






plasmidとGFP+double terminatorのdigestion

以下の組成で試薬を調整した。

Reagent Amount
DW 5.6 uL
10x H Buffer 1 uL
0.1% BSA 1 uL
Spe I 0.2 uL
Pst I 0.2 uL
plasmid 2 uL
Total 10 uL
Reagent Amount
DW 34 uL
10x M Buffer 5 uL
0.1% BSA 5 uL
Xba I 4 uL
Pst I 0.4 uL
GFP+double terminator 1.6 uL
Total 50 uL













  1. 37C,1hour置いた。
  2. Mycrocon YM-10を使って切れ端の除去を行う。sampleが500 uLになるようにTEを加え、カラムに移した。
  3. 4C,14000g,1hour遠心した。
  4. 上澄みが30 uL前後残ったので、それを新しいcollection tubeに移し、カラムを逆さまにして挿入した。
  5. 4C,1000g,3min遠心した。
  6. GFP+double terminatorが30 uL、plasmid25 uL回収できたので、各1/10量の3M CH3COONaを加えた。
  7. 100%EtOHを回収量の2.5倍量加え、voltexにかけた。
  8. 4C,15000rpm,10min遠心した。
  9. 上澄みを捨て、70%EtOHを500 uL加え、voltexにかけた。
  10. 4C,15000rpm,5min遠心した。
  11. 上澄みを捨て、真空装置へ入れよく乾燥させた。
  12. 2 uLのTEで溶かした。
  13. -20Cで凍結保存した。


Transformation of BAC Vector

  • Used DH5Alpha and MG1655 strains for electroporation
  • Plated at 19:15
  • Will be incubated for 18 h