Team:Paris/September 8

From 2008.igem.org

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(condition tested)
(Overnight ligation)
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*we obtained after elution  [dna] = 24ng/µL
*we obtained after elution  [dna] = 24ng/µL
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== Overnight ligation ==
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== Overnight ligation (16°C)==
* DNA 4 µL
* DNA 4 µL

Revision as of 16:54, 9 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

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)
  • we obtained after elution [dna] = 24ng/µL

Overnight ligation (16°C)

  • DNA 4 µL
  • 10X BUFFER 2 µL
  • LIGASE 1 µL
  • H2O 13 µL