I am using a vector that has repeat sequences, and using Mach1 competent cells. I heard that if the cells are not stable cells, then we shouldn't grow them in 37 to avoid recombination to happen between the repeat sequences.
It is fine to grow them at 30oC, they will just grow more slowly. I wouldn't use much less than 50-100ng of vector for routine cloning but there is not really an exact figure.
Dear Tiyas Sengupta
you can try to use different e.coli strain deleted for recombinase enzimes which guarantee higher stability of dna
https://www.thermofisher.com/order/catalog/product/C737303
https://www.agilent.com/en/product/mutagenesis-cloning/competent-cells-competent-cell-supplies/competent-cells-for-difficult-cloning/sure-competent-cells-233085
good luck
Manuele
Mach1 cells are very stable at 37°C, so you can grow them at 37°C. You can also use 30°C if you want, but there is no issue if you use 37°C to grow Mach1 cells. The vector: insert ratios of 1:3 and 1:5 work very effectively. You can use either one of them or both. Best wishes!
- Badges
- Science topic
- Similar topics
- Papers