SmaI cuts in both AAV ITRs and can serve as a diagnostic that the ITRs are intact.
Comments
SmaI cuts in both AAV ITRs (these can be unstable in bacteria). When culturing the bacteria containing AAV ITRs, do not culture for more than 16-18 hours at 37°C because deletions may occur especially in stationary cultures. A missing ITR can be tolerated in about 20% of a plasmid prep. SmaI cuts in both AAV ITRs and can serve as a diagnostic that the ITRs are intact. One or the other ITRs may be deleted (and so will the SmaI site) during culturing and cloning. If an ITR is deleted, then the SmaI digests will generate larger molecular weight bands.
Media
ATCC® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37°C
Propagation:
Streak a loopful of culture on an LB + 50 µg/mL ampicillin agar plate. Incubate liquid cultures at 37°C for no more than 16-18 hours. Isolate DNA using standard plasmid preparation procedures. Up to 20% of the plasmid population with one ITR deleted is tolerated
References
Burger C. et al. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system.
Molecular Therapy : 10(2):302-17, 2004 PubMed: 15294177
