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Team:GeorgiaTech/Lambda Recombineering
From 2013.igem.org
(Created page with "==Purpose== To replace the ''dsbA'' from BL21(De3) strain of E. coli ==Background== ==Starting Materials== *Vector containing Lambda Red Recombinase (Temperature Sensitive 30...") |
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First, PKD46 needs to be transformed into an electrocompetent BL21(DE3) | First, PKD46 needs to be transformed into an electrocompetent BL21(DE3) | ||
| - | This was done using | + | This was done using [[electroporation]] |
Full protocol that will be followed is shown here: [[File: BK-igem13-Recombineering (1).pdf]] | Full protocol that will be followed is shown here: [[File: BK-igem13-Recombineering (1).pdf]] | ||
Revision as of 20:37, 27 September 2013
Contents |
Purpose
To replace the dsbA from BL21(De3) strain of E. coli
Background
Starting Materials
- Vector containing Lambda Red Recombinase (Temperature Sensitive 30C , Ampicillin Resistant)
- Electrocompetent BL21 (De3) Cells
Design
First, PKD46 needs to be transformed into an electrocompetent BL21(DE3)
This was done using electroporation
Full protocol that will be followed is shown here: File:BK-igem13-Recombineering (1).pdf
Results
July 17th, 2013
Electrocompetent transformation is done in order to transform the pkd46 vector into competent bl21(DE3) cells. The transformants were plated on an ampicillin plate.
July 18th, 2013
No growth, transformation failed.
July 19th, 2013
Transformation is repeated.
July 20th, 2013
No growth on plates yet.
July 21th, 2013
Good growth on plates. PLD46 vector has successfully been transformed into the BL21(DE3) cells.
An LB + Amp media solution is made and inoculated with a single colony
July 22th, 2013
The Bl21(DE3) strain hosting the pKD46 vector is made electrocompetent, and the dsba mutant dna (kanamycin cassette) is transformed into the cells. Cells are plated on Kanamycin + LB agar plates.
July 23th, 2013
No growth on kanamycin plates. Transformation failed
