Inoculated a total of 10, 5mL 1x Marine Broth in 50mL conicals. 5 tubes received 1mL of the original culture started on 20090419. 4 tubes received 1mL of the secondary culture (from 20090421). 1 tube was inoculated with a colony from the plate streaked on 20090424. Incubated all tubes @ 28C, 200RPM. Used a higher temp. to encourage faster/more robust growth.
Bacteria – C. pugetti plate (from 20090424)
An additional yellowing has occurred on the plate. This is in accordance with the Dyksterhouse et al. paper. Colonies still barely visible.
Bacteria – C. pugetti plate (from 20090424)
There appears to be growth on the plate. There is a large, bright yellow/chartreuse region on the plate where the streaking was initiated. Additionally, upon close inspection, it appears as though colonies can be seen.
Bacteria – C. pugetti liquid culture
Results: It has now been 8 days since revival of the ATCC freeze dried culture. The media still looks a bit cloudy, but hasn’t really changed since the second or third day post-revival. Of note, there does seem to be an accumulation of clumps in the tube. These may or may not be clumps of cells. Will consult with Steven when he returns. Might need to pass cells and take some ODs to really assess changes in growth as these cells may not be as robust as E. coli or other common lab cultures.
Bacteria – C. pugetti plate
Streaked C. pugetti onto marine broth plate + a few biphenyl crystals and incubated at RT over the weekend.
Results: No growth as of Monday, 20090427.
Reverse Transcription – cDNA from DNased Abalone RNA from 20090420
Prepared cDNA using an equal amount of RNA from all samples (442.6ng). This amount was based on using the maximum allowable volume of RNA for the RT rxn AND the sample with the lowest [RNA] (08:3-20; 24.59ng/uL). cDNA was prepared according to the Promega MMLV RT recommendations. Here is the work up for the cDNA rxns. cDNA was stored @ -20C.
qPCR – Abalone DNased RNA from yesterday
Performed qPCR to evaluate gDNA removal w/ 2x Immomx and SYTO 13. qPCR/plate set up is here.
Results: The two cDNA samples come up as positive. No flourescence detected in any other gamples. However, melting curves look suspicous despite the fact that the “Quantitation” view indicates now amplification.
Bacteria – C. pugettii culture CONTINUED (from 20090419)
Transferred 1mL of the culture to a 50mL conical containing 4mL of 1x Marine Broth and a couple crystals of biphenyl. Kept the cap loosened and incubated at 20C with shaking at 200RPM. Kept the existing culture in the incubator as well. No apparent growth.
gDNA Removal – Abalone RNA from 20090402 and 20090331
Transferred 50uL of RNA to fresh tubes and processed them using the Ambion Turbo DNA-free kit according to the manufacturer’s protocol.
Bacteria – C. pugettii culture
Rehydrated ATCC isolate according to directions. Added 5mL of 1x Marine Broth to a glass culture tube. Used 1mL of this to rehydrate ATCC sample and then added back into the culture tube. Added a few crystals of biphenyl, which had been exposed to UV for ~5mins prior. Incubated at 20C, no shaking. This was done at ~11:30AM.