Features
- Fast BAC modification (2-3 weeks)
- High Red/ET Recombination efficiency
- Convenient removal of the Red/ET plasmid
- Efficiency much higher as with the pSacB-neo system
Applications
- Fragment exchange
- Marker insertion and deletion without leaving a selection marker or any unwanted sequences
- Introduction of short sequences e.g. point mutations, loxP sites, restriction sites, etc.
- Can also be used for bacterial chromosome modifications and common ColE1 origin plasmids
Overview of two-step Exchange of a Non-Selectable Gene
1. Transformation of BAC host with Red/ET
In a first step the Red/ET plasmid pSC101-BAD-gbaA is transferred into the E.coli host that contains the BAC.
2. First Red/ET step
The expression of genes mediating Red/ET is induced by adding L-arabinose and a temperature shift from 30°C to 37°C. After induction, the cells are prepared for electroporation and the PCR product (rpsL-neo PCR product counter-selection/selection cassette) with the added homology arms is electroporated.
3. Selection & Extraction of the modified BAC DNA
Selection for colonies carrying the modified BAC. Only colonies carrying successfully modified BACs will survive kanamycin selection on the agar plates. A subsequent DNA mini preparation is used to confirm the successful integration of the counter-selection/ selection cassette.
4. Second Red/ET step
The expression of genes mediating Red/ET is induced by adding L-arabinose and a temperature shift from 30°C to 37°C. After induction, the cells are prepared for electroporation. The non-selectable DNA, which can be either just an oligonucleotide harboring the right and the left homology arms of the selection cassette and a point mutation (control reaction) or a gene flanked by homology arms, will be electroporated. The rpsL-neo counter-selection/selection cassette will be replaced by the non-selectable DNA.
5. Selection for the absence of the counter-selection/selection cassette
Only colonies in which the selection/counter-selection cassette was replaced by the non- selectable DNA fragment will grow on streptomycin containing plates. A subsequent DNA mini preparation is used to confirm the successful integration of the counter-selection/ selection cassette.
Description
This is a new version of counter-selection cassette pRpsL-neo based on Streptomycin selection. The kit is designed to modify any type of bacterial artificial chromosomes (BACs) within 2-3 weeks by using a counter-selection cassette. The included counter-selection cassette pRpsL-neo is based on Streptomycin selection which shows a much higher efficiency than pSacB-neo or comparable systems.
This kit can also be used to work on bacterial chromosomes and common ColE1 origin plasmids. This Kit combines the high Red/ET efficiency plus convenient removal of the Red/ET Recombination protein expression plasmid pRedET after recombination.
Contents
- Red/ET Recombineering protein expression plasmid pRedET. Any E.coli strain can be made Red/ET proficient by transformation with this plasmid
- BAC host E.coli strain DH10B already carrying the Red/ET plasmid
- pRpsL-neomycin template to be used for your own experiments
- Positive controls to introduce a point-mutation in a 150 kb BAC
- Detailed protocols, descriptions of plasmids, maps and sequences
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