Team:HokkaidoU Japan/Notebook/August25

From 2010.igem.org

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|yes
|yes
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|-
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|1-3A 34
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] 34
|yes
|yes
|-
|-
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|1-3A 12
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|[[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] 12
|yes
|yes
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|}
<hr width="50%">
<hr width="50%">
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* 1-3A had colonies in lated on mediums containing 34 ug/uL of chloramphenicol
+
* [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] had colonies in lated on mediums containing 34 ug/uL of chloramphenicol
** So the medium and antibiotic consentration seems ok,
** So the medium and antibiotic consentration seems ok,
* Medium on which M 12 was plated was inoculated with chloramphenicol after solidification so there were some irregularities and colonies on the fringe
* Medium on which M 12 was plated was inoculated with chloramphenicol after solidification so there were some irregularities and colonies on the fringe
-
* Colonies of 1-3A and 12 were obviously different
+
* Colonies of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] and 12 were obviously different
→ So we suspected issues in Ligation and DNA concentration bused for transformation
→ So we suspected issues in Ligation and DNA concentration bused for transformation
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[[Image:HokkaidoU Japan 20100825a.jpg‎|200px|right|thumb|Electrophoresis of 1uL of 1-3A DNA]]
[[Image:HokkaidoU Japan 20100825a.jpg‎|200px|right|thumb|Electrophoresis of 1uL of 1-3A DNA]]
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*Added 1 uL of 1-3A into 30 uL of competent cells
+
*Added 1 uL of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]] into 30 uL of competent cells
*Transformation was successful  
*Transformation was successful  
-
*Electrophoresed 1 uL of 1-3A，calculated amount of transformed DNA
+
*Electrophoresed 1 uL of [[Team:HokkaidoU_Japan/Parts#BioBricks|1-3A]]，calculated amount of transformed DNA
→Estimated concentration 2 ng/uL
→Estimated concentration 2 ng/uL
<div style="clear:both"></div>
<div style="clear:both"></div>
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=pSB1C3をエタ沈で濃縮，Ligation=
+
=Ethanol precipitation of pSB1C3 and Ligation=
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===エタ沈===
+
 
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# 3 M 酢酸ナトリウム 8.2 uL加える
+
===Ethanol precipitation===
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# 100%エタノールを205 uL加え，混ぜる
+
 
-
# 15,00rpm@4℃で10分間遠心する
+
# Added 8.2 uL of sodium acetate [3 M]
-
# 上清を捨て，500 uL 70%エタノールを加え，混ぜる
+
# Added 205 uL of Ethanol [100%] and mixed
-
# 15,00rpm@4℃で5分間遠心する
+
# Centrifuge at 15000rpm, 4C for 10 min
-
# 上清を捨て，真空デシケータで乾燥させる
+
# Discarded supernatant and added 500 uL of Ethanol [70%], mixed
-
# DW 8.2 uLを加え，元の10倍に濃縮する
+
# Centrifuge at 15000rpm, 4C for 5 min
 +
# Discarded supernatant
 +
# Dried via vacuum desiccator
 +
# Added 8.2 uL of ADW
 +
* Concentration increased 10 fold
===Ligation===
===Ligation===
-
# 8.2 uL DNA solutionに同量のLigation Solutionを加える
+
# Mixed 8.2 uL of DNA solution with the same volume of Ligation Solution
-
# T4 ligase 1 uLを加え，16℃で30分間インキュベート
+
# Added extra 1 uL of T4 ligase
-
# 明日，電気泳動・ゲル抽・Transformation
+
# Incubated at 16C for 30 min
 +
Tomorrow will do aditional Electrophoresis gel extraction and transformation
 +
 
 +
=Digestion of PCR products amplified using digestion visualization primers=
 +
 
 +
 
 +
[[Image:HokkaidoU Japan 20100825c.JPG‎ |200px|right|thumb|Electrophoresis after digestion, differences in length is visible]]
 +
 
 +
Calculated the concentration of DNA amplified yesterday
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=らおりプライマーで作ったPCR productsのDigestion=
 
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[[Image:HokkaidoU Japan 20100825c.JPG‎ |200px|right|thumb|]]
 
-
前日のPCRから，増やすことのできたパーツのDNA量を求めた
 
{|
{|
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|}
|}
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===Digestion 反応系===
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===Digestion Reaction===
{|style="text-align:center;" class="protocol"
{|style="text-align:center;" class="protocol"
!Reagent
!Reagent
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|}
|}
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→37℃で60分
+
→Incubated at 37C for 60 min
-
→ラオリプライマーの真価を電気泳動で確認
+
→Confirmed how awesome new primers are

Latest revision as of 07:50, 27 October 2010

Notebook

Transformation Results

Colony Check

Plate Colonies
M 34 no
M 12 yes
I 34 no
I 34 yes
1-3A 34 yes
1-3A 12 yes
  • 1-3A had colonies in lated on mediums containing 34 ug/uL of chloramphenicol
    • So the medium and antibiotic consentration seems ok,
  • Medium on which M 12 was plated was inoculated with chloramphenicol after solidification so there were some irregularities and colonies on the fringe
  • Colonies of 1-3A and 12 were obviously different

→ So we suspected issues in Ligation and DNA concentration bused for transformation

Estimation of 1-3A Consentration

Electrophoresis of 1uL of 1-3A DNA
  • Added 1 uL of 1-3A into 30 uL of competent cells
  • Transformation was successful
  • Electrophoresed 1 uL of 1-3A，calculated amount of transformed DNA

→Estimated concentration 2 ng/uL

Ethanol precipitation of pSB1C3 and Ligation

Ethanol precipitation

  1. Added 8.2 uL of sodium acetate [3 M]
  2. Added 205 uL of Ethanol [100%] and mixed
  3. Centrifuge at 15000rpm, 4C for 10 min
  4. Discarded supernatant and added 500 uL of Ethanol [70%], mixed
  5. Centrifuge at 15000rpm, 4C for 5 min
  6. Discarded supernatant
  7. Dried via vacuum desiccator
  8. Added 8.2 uL of ADW
  • Concentration increased 10 fold

Ligation

  1. Mixed 8.2 uL of DNA solution with the same volume of Ligation Solution
  2. Added extra 1 uL of T4 ligase
  3. Incubated at 16C for 30 min

Tomorrow will do aditional Electrophoresis gel extraction and transformation

Digestion of PCR products amplified using digestion visualization primers

Electrophoresis after digestion, differences in length is visible

Calculated the concentration of DNA amplified yesterday


RBS 27 ng/uL 312 bp
GFP 54 ng/uL 1020 bp
dT 49 ng/uL 429 bp

Digestion Reaction

Reagent Amount
10x M buffer 1 uL
DW 4.5
DNA 2.5
BSA 1
EcoR I 0.5
Pst I 0.5
Total 10 uL

→Incubated at 37C for 60 min

→Confirmed how awesome new primers are
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August25"