Team:Chiba/Notebook/Calendar/29 September 2009

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([[Team:Chiba/Notebook/Calendar/28_September_2009|28_September_2009]] <|>[[Team:Chiba/Notebook/Calendar/30_September_2009|30_September_2009]])
([[Team:Chiba/Notebook/Calendar/28_September_2009|28_September_2009]] <|>[[Team:Chiba/Notebook/Calendar/30_September_2009|30_September_2009]])
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== Gel electrophoresis ==
 +
Yesterday's oparation is [https://2009.igem.org/Team:Chiba/Notebook/Calendar/28_September_2009 here].
 +
 +
 +
*Today's operation
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10:40-
 +
 +
Mini Prep.
 +
 +
 +
13:30-
 +
 +
Gel electrophoresis(135 V, 27 min)
 +
 +
 +
 +
---DNA Clean & Concentrator---
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 +
 +
 +
15:50-
 +
 +
Gel electrophoresis again(135 V, 27 min)
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 +
== Digestion Test ==
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*Sample
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Vector(plux-GFP)
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 +
Insert(sfGFP)
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 +
 +
*Vector
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<table width="100" border="1" cellpadding="0"  cellspacing="0" bordercolor="#000000"><tr>
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<td width="50">Vector DNA</td>
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<td width="50">30 &mu;L</td>
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</tr>
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<tr>
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<td>Nco1</td>
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<td>1 &mu;L</td>
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</tr>
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<tr>
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<td>BamH1</td>
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<td>1 &mu;L</td>
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</tr>
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<tr>
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<td>Buffer3</td>
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<td>5 &mu;L</td>
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</tr>
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<tr>
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<td>BSA</td>
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<td>5 &mu;L</td>
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</tr>
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<tr>
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<td>NFW</td>
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<td>8 &mu;L</td>
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</tr>
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<tr>
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<td>Total</td>
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<td>50 &mu;L</td>
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</tr>
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</table>
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*Insert
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<table width="100" border="1" cellpadding="0"  cellspacing="0" bordercolor="#000000"><tr>
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<td width="50">Insert DNA</td>
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<td width="50">20 &mu;L</td>
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</tr>
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<tr>
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<td>Nco1</td>
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<td>1 &mu;L</td>
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</tr>
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<tr>
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<td>BamH1</td>
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<td>1 &mu;L</td>
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</tr>
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<tr>
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<td>Buffer3</td>
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<td>3 &mu;L</td>
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</tr>
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<tr>
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<td>BSA</td>
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<td>3 &mu;L</td>
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</tr>
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<tr>
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<td>NFW</td>
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<td>2 &mu;L</td>
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</tr>
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<tr>
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<td>Total</td>
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<td>30 &mu;L</td>
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</tr>
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</table>
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18:55-
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Gel electrophoresis(135 V, 25 min)
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 +
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19:45
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Took a picture
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== Transformation ==
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*Sample
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Competent Cells : XL10G(K)
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 +
Plasmid : plux-GFP(Cm)
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21:45-
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CmのプレートにまいてCultured it at 37 degrees Celsius.
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 +
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== Digestion ==
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*Sample
 +
Vector : plux-GFP(p15A)
 +
 +
Insert : sfGFP
 +
 +
Enzymes : Nco1, BamH1
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 +
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21:17
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We added Enzymes and took it 37 degrees Celsius.
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 +
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---3 h---
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We took it 65 degrees Celsius for 酵素を失活させる
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 +
 +
---20 min---
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24:42
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We kept it in refrigerator.
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== PCR ==
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*Template
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plux-HSUTK
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 +
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*Element of mixtures
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 +
<table width="200" border="1" cellpadding="0"  cellspacing="0" bordercolor="#000000"><tr>
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<td width="100">Template</td>
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<td width="100">1 &mu;L</td>
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</tr>
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<tr>
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<td>Primer(F)</td>
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<td>2.5 &mu;L</td>
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</tr>
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<tr>
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<td>Primer(R)</td>
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<td>2.5 &mu;L</td>
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</tr>
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<tr>
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<td>Buffer(x 10)</td>
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<td>5 &mu;L</td>
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</tr>
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<tr>
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<td>dNTP</td>
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<td>4 &mu;L</td>
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</tr>
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<tr>
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<td>DNA pol.</td>
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<td>1 &mu;L</td>
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</tr>
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<tr>
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<td>dH<sub>2</sub>O</td>
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<td>34 &mu;L</td>
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</tr>
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<tr>
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<td>Total</td>
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<td>50 &mu;L</td>
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</tr>
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</table>

Latest revision as of 08:00, 3 October 2009

>Go to the Notebook page

(28_September_2009 <|>30_September_2009)



Contents

Gel electrophoresis

Yesterday's oparation is here.


  • Today's operation

10:40-

Mini Prep.


13:30-

Gel electrophoresis(135 V, 27 min)


---DNA Clean & Concentrator---


15:50-

Gel electrophoresis again(135 V, 27 min)

Digestion Test

  • Sample

Vector(plux-GFP)

Insert(sfGFP)


  • Vector
Vector DNA 30 μL
Nco1 1 μL
BamH1 1 μL
Buffer3 5 μL
BSA 5 μL
NFW 8 μL
Total 50 μL


  • Insert
Insert DNA 20 μL
Nco1 1 μL
BamH1 1 μL
Buffer3 3 μL
BSA 3 μL
NFW 2 μL
Total 30 μL


18:55-

Gel electrophoresis(135 V, 25 min)


19:45

Took a picture

Transformation

  • Sample

Competent Cells : XL10G(K)

Plasmid : plux-GFP(Cm)


21:45-

CmのプレートにまいてCultured it at 37 degrees Celsius.



Digestion

  • Sample

Vector : plux-GFP(p15A)

Insert : sfGFP

Enzymes : Nco1, BamH1


21:17

We added Enzymes and took it 37 degrees Celsius.


---3 h---


We took it 65 degrees Celsius for 酵素を失活させる


---20 min---

24:42

We kept it in refrigerator.



PCR

  • Template

plux-HSUTK


  • Element of mixtures
Template 1 μL
Primer(F) 2.5 μL
Primer(R) 2.5 μL
Buffer(x 10) 5 μL
dNTP 4 μL
DNA pol. 1 μL
dH2O 34 μL
Total 50 μL
Retrieved from "http://2009.igem.org/Team:Chiba/Notebook/Calendar/29_September_2009"