Team:Paris/September 8

From 2008.igem.org

(Difference between revisions)
(Overnight ligation (16°C))
(Digestion)
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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==

Revision as of 14:30, 12 September 2008

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Contents

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 gel excision
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