Hello,
I am currently working on generating deletional mutants in Agrobacterium C58 using a construct containing upstream and downstream fragments of a target gene. The construct also includes a spectinomycin resistance gene for selection.
After performing electroporation and spreading the transformants on spectinomycin plates, I conducted colony PCR using primers located inside the upstream and downstream fragments to screen for successful integrants.
However, I only obtained a single band in the colony PCR corresponding to the deletional construct, and not the expected two bands (wild-type gene allele and integrant with the construct).
I am seeking advice from researchers who have experience with a similar process. Could you kindly share any insights on the following:
1. Potential reasons for obtaining a single band in the colony PCR instead of two bands (wild-type allele and integrant).
2. Tips for improving the efficiency of obtaining transformants with the deletional construct.
3. Construct design considerations that could impact the success of the first crossover event.
Any additional thoughts or troubleshooting steps that could help in successfully generating deletional mutants in Agrobacterium C58 would be greatly appreciated.
If the Spectinomycin cassette flanked by upstream and downstream DNA is linear (a PCR product or a restriction fragment) then you can only generate a replacement (ie double recombination event) so you should only see the deletion product.
However if you are introducing it as a circular plasmid then you could obtain single recombination events with both the deletion and the wt alleles.
Use CRISPR to introduce a double strand break near your integration site. This will stimulate homologous recombination.
Using longer homology arms generally increases the probability of insertion.
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