Team:Arizona State/Notebook/Ernesto
Notebook:Ernesto Luna
LuxI Transformation
Thursday, 6/9/16
- Gathered 2 amp agar plates from the cold room (4C) and warmed them up in the incubator at 37.C.
- Prepared an ice bath to thaw out the frozen cells and maintain the plasmid DNA cold.
- Gathered 1 tube of BL21 cells from -80C storage and placed in the ice bath to thaw (~10 min).
- Gathered "tube 2" of concentrated LuxI plasmid from the refrigirator and placed in the ice bath.
- Prepped 2 eppendorf tubes (1.5mL), one for the LuxI transformation, and another for a control. Mixed as follows and kept on ice for 10 mins.
- Took warm agar plates (2) from the incubator and labeled with "plasmid (or negative control), cell strain, Abx, initials, date".
- Pipetted entire volume of each mixture onto each respective plate.
- Added ~7-10 glass innoculation beads to each plate and swirled for a minute.
- Disposed of innoculation beads in flask containing EtOH.
- Placed plates upside down in the incubator and left over night.
| A | B | |
| 1 | LuxI Transformation | LuxI Neg. (Control) |
| 2 | 8uL dH20 2uL plasmid 40uL BL21 cells | 10uL dH20 40uL BL21 cells |
LuxI Transformation
Friday, 6/10/16
Followed up to check for growth. 3 small cultures grew in the LuxI plate and displayed a faint,red color. Disposed of negative control plate (no growth), and parafilmed LuxI plate to store in the cold room (4C).
Receiver Transformation in DH5AT
Monday, 9/19
Inoculated and grew cultures for each of the senders that I will be using to induce F2620. Due to well amount limitations on the 96 well plate, we are only inducing with 4 senders at a time. In this first run, we are only inducing with AubI, LuxI, LasI, and EsaI media. Below is a table of the 96 well plate organization (scroll to the sides to see everything). Intended to run the 8hr induction tomorrow, but I read Cassandra's protocol wrong and forgot to grow receiver cultures for seeding. Will be doing everything in triplicate and will use sender media concentrations of 10% and 50% in well volumes of 200uL. It is important to note that all senders are in Bl21 and all receivers are in DH5aT.
