I inserted my D-product artificial-miRNA into pDONR207 DH5alpha, colony confirmed with PrimerA , PrimerB also with attB1, and attB2 primers. similarly, the plasmid was confirmed with the same primers. but after the LR reaction, I tried to do colony PCR with the same condition as the BP reaction. but I am not getting any results. My previous two constructs were cloned with the above method
I think you mean a PCR made from a bacterial colony stuck with a toothpick on a petri dish ?
If that's what you're talking about, the colony-PCR can be tricky. When the bacterial colony is taken from the Petri dish, care should be taken not to poke into the agar, because the agar contains taq-polymerase inhibitors, and also diverse enzyme inhibitors... We can also replace the agar with agarose, in the Luria Bertani medium, which could avoid this inhibition of PCR.
Best wishes.
Gibb AP, Wong S. Inhibition of PCR by agar from bacteriological transport media. J Clin Microbiol. 1998 Jan;36(1):275-6. doi: 10.1128/JCM.36.1.275-276.1998. PMID: 9431965; PMCID: PMC124852.
Thank you for help, I repeated the experiment and got positive results.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning