Tag Archives: Cycloclasticus pugetii

PCR – C.pugetti DNA from 20090513 & 20090526

Performed PCR as set up here using universal bacterial 16s primers (sequences provided by Sara Kelly).

Lane 1 – 100bp ladder

Lane 2- 5/13 DNA

Lane 3 – 5/26 DNA

Lane 4 – H2O

Results: Contamination in the water sample. Hopefully this is just bad technique (never thought I’d say that about myself) and not general, bacterial contamination in the primer stocks, since the stocks (nor the PCR rxns) were prepared steriley. Will repeat.

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DNA Gel – JGI QC check of C. pugetti DNA from 20090526

Lane 1 – 15ng standard (5uL)

Lane 2 – 31ng standard (5uL)

Lane 3 – 63ng standard (5uL)

Lane 4 – Marker 2 (5uL)

Lane 5 – C. pugetti DNA (5uL: 4uL + 1uL 5x dye)

Lane 6 – Marker 3 (5uL)

Lane 7 – 125ng standard

Lane 8 – 250ng standard (5uL)

Lane 9 – 500ng standard (5uL)

Results: Looks great! Will run PCR using universal 16s primers for sequencing.

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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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gDNA Isolation – C.pugetti

Isolated according to JGI protocol (Word doc). Used 100mL, 8 day old culture inoculated from a plate on 20090505. Resuspended pellets in 740uL of TE and took an OD600 via the NanoDrop. Diluted the sample appropriately to an OD600 ~ = 1.0 in a final volume of 740uL TE (see the last three measurements for OD600 of final dilution).

Followed protocol. Recovered 300uL of aqueous phase prior to precipitation with isopropanol (Step #21). Resuspended DNA in 20uL of H2O. Will run samples on gel according to JGI instructions.

Lane 1 – 15ng standard (5uL)

Lane 2 – 31ng standard (5uL)

Lane 3 – 63ng standard (5uL)

Lane 4 – Marker 2 (5uL)

Lane 5 – C. pugetti DNA (5uL: 3uL + 2uL dye)

Lane 6 – Marker 3 (5uL)

Lane 7 – 125ng standard

Lane 8 – 250ng standard (5uL)

Lane 9 – 500ng standard (5uL)

Results: DNA looks stellar! Just like the example gel in the JGI QC documentation. however, looks to be too little TOTAL yield of DNA to send for sequencing (need

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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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