Inoculated 4 x 5mL 1xLB + Kan50 with a colony from each set of clones, incubated 37C, 200RPM, O/N. Will mini prep and send for sequencing tomorrow.
Inoculated 5mL of 1x LB + Kan50 (made by Steven on 3/23/11). Incubated O/N at 37C, 250RPM. Will perform mini preps tomorrow. The following samples were selected:
- MM09 – #1, 2, 8
- MM11 660bp – #1, 2, 8
- MM10 2/8/11 – #1, 2
- MM04 1/19/11 – #2, 3
- MM11 3000bp – #3
- MM04 1500bp – #4
- MM06 1/19/11 – #1, 2
- MM04 550bp – #1, 2
- MM05 1/19/11 – #1, 2
Mini prepped 3mLs of each culture, according to Qiagen protocol. Samples were eluted with 50uL of Buffer EB.
Inoculated 5mL of 1x LB + Kan50 (made by Steven on 3/23/11). Incubated O/N @ 37C, 250RPM. Will perform mini preps tomorrow. The following samples were selected (red text on the plate):
400mL O/N culture (1x LB+1% NaCl, 37C, 150RPM, 1L flask) of V.vulnificus (STRAIN??) and V.tubiashii (Strain: RE22) were pelleted (4300RPM, 25C, Sorvall ST-H750 rotor). Supe was removed and pellets were each resuspended in 50mL sea water. 1mL was taken from each to use for dilutions to determine colony forming units (CFU).
Two containers were set up with each containing 16 C.gigas, and air stone and 8L of sea water. The entire 50mL of V.vulnificus was added to one of the containers. 8 oysters were sampled (gill and mantle tissue) from each container at 1hr and 3hrs after the addition of V.vulnificus culture and immediately frozen on dry ice. Samples were stored @ -80C in the “Gigas Vibrio Exposure 1,3hrs 1/11/11″ box. Additionally, 1mL samples of the water were taken at each time to determine CFU in the water.
In addition to the samples taken above, the following tissues were taken from 5 control oysters at the 3hr time point and treated/stored in the same fashion as the others, specifically for assessment of cyclooxygenase tissue distribution analysis: muscle, digestive gland/gonad (difficult to differentiate)
All oysters were measured. Morphometric data is here.
1L liquid culture (grown for 10 days) was split into two UV-sterilized bottles. Cells were pelleted in a Sorvall T21 centrifuge using the bucket rotor for 30mins @ 4200RPM, 4C. Supe was removed and bacterial pellets were stored @ -20C.
Inoculated 3 x 5mL Marine Broth + biphenyl crystals (UV sterilized) cultures from frozen stock in 50mL Falcon tubes. Incubated 200RPM, 28C. Will use these are starter cultures to inoculate larger cultures.
Inoculated 1x 1L of MB + biphenyl crystals from frozen stock. Incubated 28C, 200RPM. MB was autoclaved with the biphenyl crystals.