Tag Archives: bacterial culture

gDNA Isolation – C. pugetti (from 20090518)

Followed JGI “Bacterial genomic DNA isolation using CTAB” protocol(Word doc) with the following notes/changes.

Two, 1L cultures were transferred to 4 total jars (~500mL in each jar) for centrifugation. Jars had been briefly washed with 95% EtOH prior to use.

Cells were pelleted in Sorval T21 using a bucket rotor for 30mins., 4200RPM, 25C.

Supe was removed and all four pellets were resuspended with a total of 10mL of TE.

OD600 = ~2.1, so added an additional 10mL of TE.

OD600 = ~1.2, so proceeded with procedure.

Due to volume of the prep, the sample was split into two, 50mL centrifuge tubes (washed with 95% EtOH prior to use) prior to the addition of 5M NaCl.

Disaster strikes! Turns out the tubes being used were not resistant to chloroform. This was realized after spinning at 18,000RPM, 25C 10mins in a Sorval SL-50T rotor in the Sorval T21 centrifuge.

However…

I recovered the aqueous phase anyway, as it was still contained in the tube and had no contact with the rotor surface(s). Due to a subsequent phenol:chloroform:IAA step, I split the aqueous phase into 24 x 1.5mL snap cap tubes and proceeded according to protocol. The final DNA pellet, prior to resuspension were combined from all the tubes into a single 1.5mL snap cap tube in a final volume of 400uL of TE.

Results: DNA from today looks good with excellent yield. DNA from 20090513 doesn’t look as nice AND the concentration doesn’t jive with the QC gel that was run on 20090513. Will run out on gel according to JGI protocol to evaluate quality further.

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Bacteria – C. pugetti liquid cultures

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.

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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.

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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.

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