I am knocking in a DNA fragment with homology to the E. coli genome via a lambda red knock-in protocol.
I induce the cells to express lambda red genes during competent cell preparation. The lambda red genes are on a plasmid that has antibiotic resistance X.
I introduce my DNA fragment with antibiotic resistance Y via electroporation. Then, I recover for 1 hour in plain media after electroporation and then plate my cells.
At what point after electroporation does the actual homologous recombination take place? When is it ok for the cells to lose the plasmid with the lambda red genes? Can I plate on just antibiotic Y or should I plate on both antibiotics X and Y? Thanks.
You should only plate on antibiotic Y. The induction produces more that enough protein for the recombination. Just a word of advice the 1 hour recovery is rarely enough (atleast in my hands) you should allow overnight recovery in SOC at room temp without shaking then plate on antibiotic Y at 37C to cure the lamda red plasmid.
I agree with Hanna Alalam, the recombination event usually takes place soon upon introduction of the DNA fragment. The recombination proteins have already been produced when you expressed the lambda red genes. You should not use antibiotic X after the transformation.
Hello
I am going to insert 4500bp gene construct into mouse genome by homologous recombination. my homologous arms are about 2kb each side which are the normal genome sequence in which we are going to insert about 200bp in to our gene and mutant it.
first of all I want to know without crispar or TALEN can it be possible ?
the size of construct is too much can it cause problem?
we inject in pronucleus by micro injection but I wonder does it go under splicing and introns add it as we collect just exon region for recombination
I didnt get the result as I have no GFP or marker for prove it but PCR the Blast
please help me find the solution or my problem in recombination
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA