Team:HokkaidoU Japan/Notebook/September24

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*Miniprep of Arac+RBS+pSB1A3
 +
*Follow quality check
 +
 +
= Miniprep of Arac+RBS+pSB1A3  =
 +
 +
#Transfered E.coli solution to 1.5 mL tubes, 1 mL each
 +
#Centrifuged at 4C, 15000 rpm for 1 min
 +
#Discarded the supernatant
 +
#Suspended on 125 uL of Buffer P1 each
 +
#Added 175 uL Buffer N3 each mixed by inversion
 +
#Centrifuged at 4C, 13000 rpm for 10 min
 +
#Transfered the supernatant to filtration column
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
#Discarded the flow-through
 +
#added 500 uL of Buffer PB to filtration column
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
#Discarded the flow-through centrifuged for 1min to remove remaining buffer
 +
#Transfered filtration column to a new 1.5 ml tube
 +
#Resuspended on 50 ul of TE and incubated at RT for 1min
 +
#Centrifuged at 4C, 13000 rpm for 1 min
 +
 +
 +
== Electrophoresis of minipreped samples ==
 +
#Mixed 1 uL of sample with loading　buffer 1 uL
 +
#Added 6 uL　of λ/Hind Ⅲ　EcoR Marker
 +
#Electrophoresed
 +
 +
 +
[[Image:HokkaidoU Japan 20100924a.JPG‎|200px|left|thumb|Electrophoresis of minipreped samples]]
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 +
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
 +
 +
<br><br><br><br><br>
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 +
 +
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 +
 +
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= Digestion of plasmid and GFP+double terminator =
 +
Digestion mix According to the table below
 +
 +
{|style="text-align: center ;float:left;" class="protocol"
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!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
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''10 uL'''
 +
|}
 +
{|style="text-align: center; float:left;" class="protocol"
 +
!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
 +
|-
 +
|style="border-top:1px solid #996"|'''Total'''
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|style="border-top:1px solid #996"|'''50 uL'''
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|}
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 +
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<br><br><br><br><br><br><br><br><br><br><br><br>
 +
#incubated at 37C for 60 min
 +
#purified samples with Mycrocon YM-10
 +
#sampleが500 uLになるようにTEを加え、カラムに移した。
 +
#centrifuged at 4C,14000 G for 1h
 +
#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。
 +
#centrifuged at 4C,1000 G for 3min
 +
#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。
 +
#100%EtOHを回収量の2.5倍量加え、voltexにかけた。
 +
#centrifuged at 4C,15000 rpm for 10min
 +
#上澄みを捨て、70%EtOHを500 uL加え、voltexにかけた。
 +
#centrifuged at 4C,15000rpm for 5min
 +
#上澄みを捨て、真空装置へ入れよく乾燥させた。
 +
#2 uLのTEで溶かした。
 +
#-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

Latest revision as of 16:19, 27 October 2010

Notebook
  • 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









Digestion of plasmid and GFP+double terminator

Digestion mix According to the table below

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. incubated at 37C for 60 min
  2. purified samples with Mycrocon YM-10
  3. sampleが500 uLになるようにTEを加え、カラムに移した。
  4. centrifuged at 4C,14000 G for 1h
  5. 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。
  6. centrifuged at 4C,1000 G for 3min
  7. 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。
  8. 100%EtOHを回収量の2.5倍量加え、voltexにかけた。
  9. centrifuged at 4C,15000 rpm for 10min
  10. 上澄みを捨て、70%EtOHを500 uL加え、voltexにかけた。
  11. centrifuged at 4C,15000rpm for 5min
  12. 上澄みを捨て、真空装置へ入れよく乾燥させた。
  13. 2 uLのTEで溶かした。
  14. -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
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