Protocol 3: Growing liquid cultures of Agrobacterium prior to transformation day
Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin
Abstract
Overnight liquid cultures of Agrobacterium transformed with the plasmid(s) of interest must be prepared the night before Spizellomyces transformation day. Liquid cultures must be inoculated from active plates/colonie. If you would like to use 25% glycerol stocks of previously transformed Agrobacterium , streak the desired cells onto the appropriate LB+selection plate, let it grow for 2-4 days at 28℃, then use this plate to inoculate the liquid media at least 12 hours before the intended transformation time. Ensure proper sterile technique throughout the protocol, using either a laminar flow hood or working in the sterile area around an open flame
Before start
Perform this protocol the night before your intended Spizellomyces transformation time.
Attachments
Steps
Steps
Fill the appropriate amount of culture tubes each with 5mL of LB liquid media.
Using an inoculation loop or pipette tip, gently scrape the surface of the appropriate Agro plate to collect the bacteria or pick the colony of interest.
Swirl the loop or tip to dislodge the bacteria into the media of the appropriate culture tube.
Incubate at 28°C, shaking at 225rpm, or .
