Paris/August 3

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(Difference between revisions)
(DGAT biobrick: coR1-Pst1 digestion)
(DGAT biobrick: coR1-Pst1 digestion)
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- EcoR1 enzyme: 1µL<br>
- EcoR1 enzyme: 1µL<br>
- Pst-1 enzyme: 1µL<br>
- Pst-1 enzyme: 1µL<br>
-
- ddH<sub>/2<sub><br>
+
- ddH<sub>2<sub>O<br>
== TOPO cloning ==
== TOPO cloning ==

Revision as of 10:52, 6 August 2007

DGAT biobrick: coR1-Pst1 digestion

DGATbbdigEcoR1Pst108032007.jpg

The double digestion allows us to insert the biobrick into biobrick plasmid.
The Pst1 digestion allows us to see if the mutagenesis worked (dgat gene not digested).

  • Material:

- DNA (PCR product DGAT deltaPst-1 biobrick: see [[): 200ng = 20µL
- BSA: 0.5µL
- NEBuffer EcoR1: 5µL
- EcoR1 enzyme: 1µL
- Pst-1 enzyme: 1µL
- ddH2O

TOPO cloning

Cloning using TOPO protocol of the following purification of PCR products

Number Name Oligo F Oligo R Matrix Length Comments
P6 Lox66-DapASubtilis O8 Lox66-DapASubt-F O9 DapASubt-R B.Subtilis 168 979
P7 Lox71-FtsZ O1 Lox71-FtsZ-F O2 FtsZ-R MG1655 1260 EcoRI site in FtsZ not mutated
P8 RBS-DapAColi O20 RBS-DapAColi-F O7 DapAColi-R MG1655 952 B0030-DapAColi