Team:Paris/September 8

From 2008.igem.org

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{{Paris/Calendar_Links|September 7|September 9}}
{{Paris/Calendar_Links|September 7|September 9}}
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=Constructions=
 +
==Results of the transformation, we did yesterday==
 +
{|border="1" style="text-align: center"
 +
|'''Ligation n°'''
 +
|'''Insert'''
 +
|'''Vector'''
 +
|'''Construction'''
 +
|'''Antibio'''
 +
|'''Number of colonies'''
 +
|-
 +
|positive control
 +
|
 +
|
 +
|puc 19
 +
|Amp
 +
| +++
 +
|-
 +
|L173
 +
|D112
 +
|D187
 +
|rbs-tetR-mRFP-LVA+
 +
|Amp
 +
|0
 +
|-
 +
|L183
 +
|D134
 +
|D187
 +
| pFlhB - mRFP LVA+
 +
|Amp
 +
|0
 +
|-
 +
|L184
 +
|D149
 +
|D198
 +
| pFliL - ECFP LVA+
 +
|Amp
 +
|0
 +
|-
 +
|L185
 +
|D149
 +
|D199
 +
| pFliL - ECFP LVA-
 +
|Amp
 +
|0
 +
|}
 +
 +
=>This is a supplementary time that the experiment of ligation-transformation don't work.<br> It means that on of this step have a mistake. <br> So we need to do several test to understand where the problem is located.<br> We Hypothesis that the problem is due to a defectious Ligase.
 +
=Checking our ligases=
=Checking our ligases=
Since our ligation experiments didn't work during these few days, we presume that our problem came from the ligases. That's why we decided to check the activity of the ligases in our possession.
Since our ligation experiments didn't work during these few days, we presume that our problem came from the ligases. That's why we decided to check the activity of the ligases in our possession.
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* we will digest a plasmid with only one restriction enzyme (EcoRI).
* we will digest a plasmid with only one restriction enzyme (EcoRI).
* then we will try to ligate it back using our ligases.
* then we will try to ligate it back using our ligases.
 +
==Condition tested ==
==Condition tested ==
-
[[Image:KR000307.JPG|thumb|Before gel excision]]
+
[[Image:KR000307.JPG|thumb|Before gel excision <br> well n°4: undigested plasmid (MP101.1) <br> well °6, 7, 8, 9: digested plasmid (D202)]]
-
[[Image:KR000308.JPG|thumb|After gel excision]]
+
[[Image:KR000309.JPG|thumb|After purification]]
[[Image:KR000309.JPG|thumb|After purification]]
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==Digestion==
==Digestion==
-
reaction mixture
+
Reaction mixture
(carried  out four times )
(carried  out four times )
-
* ADN (MP101.1) 3.8 µL  
+
* ADN (MP101.1): 3.8 µL  
-
* H2O 21.9 µL
+
* H2O: 21.9 µL
-
* 10X Buffer 3 µL
+
* 10X Buffer: 3 µL
-
* BSA 0.3 µl
+
* BSA: 0.3 µl
-
* EcoRI 1 µL
+
* EcoRI: 1 µL
==Purification==
==Purification==
*We purifed our digestion products by gel extraction (Qiagen kit)
*We purifed our digestion products by gel extraction (Qiagen kit)
-
*we obtained after elution  [dna] = 24ng/µL
+
*After elution, we obtained [DNA] ~ 25 ng/µL (based on the intensity of the band)
== Overnight ligation (16°C)==
== Overnight ligation (16°C)==
-
* DNA 4 µL
+
* DNA: 4 µL
-
* 10X BUFFER 2 µL
+
* 10X Buffer: 2 µL
-
* LIGASE 1 µL
+
* Ligase: 1 µL
-
* H2O 13 µL
+
* H2O: 13 µL

Latest revision as of 19:31, 16 September 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

Constructions

Results of the transformation, we did yesterday

Ligation n° Insert Vector Construction Antibio Number of colonies
positive control puc 19 Amp +++
L173 D112 D187 rbs-tetR-mRFP-LVA+ Amp 0
L183 D134 D187 pFlhB - mRFP LVA+ Amp 0
L184 D149 D198 pFliL - ECFP LVA+ Amp 0
L185 D149 D199 pFliL - ECFP LVA- Amp 0 
=>This is a supplementary time that the experiment of ligation-transformation don't work.
 It means that on of this step have a mistake. 
 So we need to do several test to understand where the problem is located.
 We Hypothesis that the problem is due to a defectious Ligase.

Checking our ligases

Since our ligation experiments didn't work during these few days, we presume that our problem came from the ligases. That's why we decided to check the activity of the ligases in our possession. To do so:

  • we will digest a plasmid with only one restriction enzyme (EcoRI).
  • then we will try to ligate it back using our ligases.

Condition tested

Before gel excision
well n°4: undigested plasmid (MP101.1)
well °6, 7, 8, 9: digested plasmid (D202)
After purification
ligase incubation time amount of enzyme
L1 (ligase 1) 2h00 2 U (unit)
L1 2h00 400 U
L1 O/N 2 U
L1 O/N 400 U
L2 (ligase 2) 2h00 2 U
L2 2h00 400 U
L2 O/N 2 U
L2 O/N 400 U

Digestion

Reaction mixture (carried out four times )

  • ADN (MP101.1): 3.8 µL
  • H2O: 21.9 µL
  • 10X Buffer: 3 µL
  • BSA: 0.3 µl
  • EcoRI: 1 µL

Purification

  • We purifed our digestion products by gel extraction (Qiagen kit)
  • After elution, we obtained [DNA] ~ 25 ng/µL (based on the intensity of the band)

Overnight ligation (16°C)

  • DNA: 4 µL
  • 10X Buffer: 2 µL
  • Ligase: 1 µL
  • H2O: 13 µL
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