Team:HokkaidoU Japan/Notebook/August23

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=pSB1C3の活性評価=
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=pSB1C3 Activity Check=
==EcoR I/Pst I Digestion==
==EcoR I/Pst I Digestion==
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{| class="protocol"
{| class="protocol"
|'''Reagent'''
|'''Reagent'''
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|1 uL
|1 uL
|-
|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #000;"|'''70 uL'''
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|style="border-top:1px solid #996;"|'''70 uL'''
|}
|}
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* 37℃，60 min
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* Incubated at 37C for 60 min
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* 15 uL 6x Sample Bufferを加え，６レーンに分けて泳動
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* Added 15 uL of 6x Sample Buffer and electrophoresed on six lanes
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* ゲル抽出
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* Extracted from gel
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===Ligated===
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*Used 2 uL of from 100 uL of gel extracted DNA for liagation
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** Namely added 2 uL of Ligation Solution to 2 uL of DNA
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*Incubated at 16C for 30 min
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===Ligation Negative Control===
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Used 2 uL of from 100 uL of gel extracted DNA for [[Team:HokkaidoU_Japan/Protocols|Transformation]]
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=Vector PCR: We meet again!=
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===Ligationあり===
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[[Image:HokkaidoU Japan 20100823a.JPG‎|200px|right|thumb|Electrophoresis to check if PCR was succesful]]
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ゲル抽して得た100 uL DNAのうち2 uLを使用してTransformation
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===Ligationなし===
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Amplified pSB1A3, pSB1C3 and pSB1T3 by PCR as listed in the table bellow.
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ゲル抽して得た100 uL DNAのうち2 uLに2 uL Ligation Solutionを加える<br>
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16℃で30分おき，計4 uLをTransformation
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=VectorのPCRリベンジ=
 
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[[Image:HokkaidoU Japan 20100823a.JPG‎|200px|right|thumb|]]
 
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pSB1A3, pSB1C3, pSB1T3それぞれについて以下の反応系でPCRを行った
 
{| class="protocol"
{| class="protocol"
|'''Reagent'''
|'''Reagent'''
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|1
|1
|-
|-
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|style="border-top:1px solid #000;"|'''Total'''
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #000;"|'''50 uL'''
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|style="border-top:1px solid #996;"|'''50 uL'''
|}
|}
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* 前回と違い，Extentionを120 secで行った
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* Only deviation from protocol was increase to extention to 120 sec

Latest revision as of 08:30, 27 October 2010

Notebook

pSB1C3 Activity Check

EcoR I/Pst I Digestion

Reagent Amount
pSB1C3 50 uL
DW 4 uL
0.1% BSA 7 uL
10x M Buffer 7 uL
EcoR I 1 uL
Pst I 1 uL
Total 70 uL
  • Incubated at 37C for 60 min
  • Added 15 uL of 6x Sample Buffer and electrophoresed on six lanes
  • Extracted from gel

Ligated

  • Used 2 uL of from 100 uL of gel extracted DNA for liagation
    • Namely added 2 uL of Ligation Solution to 2 uL of DNA
  • Incubated at 16C for 30 min

Ligation Negative Control

Used 2 uL of from 100 uL of gel extracted DNA for Transformation

Vector PCR: We meet again!

Electrophoresis to check if PCR was succesful

Amplified pSB1A3, pSB1C3 and pSB1T3 by PCR as listed in the table bellow.

Reagent Amount
Vector 1
DW 33
10x Buffer 5
2 M 4dNTPs 5
25 mM MgSO4 3
Suffix-F 1
Prefix-R 1
KOD 1
Total 50 uL
  • Only deviation from protocol was increase to extention to 120 sec
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