Sample was removed from -20C and spun @ 4C, 16,000 x g for 30mins. Supe removed, pellet washed with 1mL 70% EtOH and spun @ 4C, 16,000 x g for 15mins. Supe removed, tube spun briefly and remainder of EtOH removed. Pellet was resuspended in 100uL of 1x TE and spec’d. Sample will be run on a gel according to JGI instructions.

Results:

The only thing that could be worse about this gel would be no sample DNA. However, what we see here (the giants smear in middle of the gel is our sample) is completely degraded OR sheared gDNA. That means this gDNA is absolutely useless now. Will start growing more cultures for another massive gDNA isolation.

Precipitate DNA from 20090526 to concentrate and run on gel for resubmission to JGI to see what happens. Added 0.1 volume of 3M sodium acetate, pH = 5.2 (40uL) and 2 volumes of 100% EtOH (880uL). Mixed throughly and incubated O/N @ -20C.

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.

One of the three starter liquid cultures from 20090706 wee used to inoculate 1L of Marine Broth + biphenyl. Incubated 200RPM @ 28C.

A recent email from JGI indicates that they are satisfied with the quality of DNA (as seen on 20090601), however their estimate of the gDNA concentration (42ng/uL) means that we have ~16ug of DNA. They requested 50ug. Based on the gel, their calculations are reasonable. However, the NanoDrop suggests that are sample is ~1350ng/uL! So, I’ve respec’d the sample and did a few dilutions to see how it looked.

Results: The undiluted sample is approximately the same concentration as initially reported. A 1:1 dilution produces the expected concentration of half the undiluted. The 1:10 and 1:100 dilutions deviate a bit from the expected concentrations, but are reasonably close. I still don’t know how to explain the discrepancy between what the gel analysis suggests vs. the NanoDrop spectrophotometric data.

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.

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.

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