I am trying to tag the C terminus of endogenous genes CDC15 and 24 (both genes neccessary for growth) with mScarlet. I transformed a PCR product with the following sequence: 40bp homology-mScarlet-Cyc1terminator-LeuLocus-40bp homology. The homology regions targeted the very end of CDC15 and 24. After transformation, I had around 50-100 colonies grow on a -Leu plate for both genes. I restreaked 8 from both on another -Leu plate. I then ran a colony PCR using Phusion polymerase on these strains, amplifying a region that covered slightly upstream and downstream of the end of the CDC genes. For all strains, I got a 500bp product, which corresponds to the tag not begin integrated at the end of the CDC gene. If it was integrated, the product would be ~3kb. What is the most likely explanation for this? Could the transformed PCR product integrated somewhere else? Is 40bp homology not enough? Could there be an issue with the colony PCR? If anyone has any insight, I'd appreciate it.
Hi there,
Either Cter tagging of these essential proteins is affecting their activity but it seems to me that cdc15 in Cter fusion is still functional... Or you should screen more clones.
Do you plate transformation mix directly onto selective medium or do you plate it first on non selective and then replica plate onto -leu plates? Sometimes selection pressure is not in favor of the expected HR event.
To answer your questions: insert is obviously inserting somewhere else, 40bp is enough for HR, your PCR is OK as you get the band corresponding to a negative result.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line