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.
Used BB & DH samples #11-17 for procedure. Followed Epigentek’s protocol. My calcs for dilutions/solutions needed are here. All solutions were made fresh before using, except for Diluted GU1 which was made at the beginning of the procedure and stored on ice in a 50mL conical wrapped in aluminum foil. Used 100ng total (50ng/uL) of each sample gDNA. No standards for a standard curve based on speaking with Mac.
| WELL | SAMPLE | WELL | SAMPLE |
| A01 | BB11 | A02 | DH11 |
| B01 | BB12 | B02 | DH12 |
| C01 | BB13 | C02 | DH13 |
| D01 | BB14 | D02 | DH14 |
| E01 | BB15 | E02 | DH15 |
| F01 | BB16 | F02 | DH16 |
| G01 | BB17 | G02 | DH17 |
| H01 | Pos. Control | H02 | Blank |
Results: Above is the graph of the results. Although it’s only a small difference between the two sites, it is statistically significant. The calcs for this graph can be found here (Excel file). It should be noted that this graph was generated using estimated values from the standard curve provided in the manufacturer’s protocol. This was done because 1) I did not run standards to generate my own curve and 2) calculating the “% methylation” not using the formula that utilizes the standard curve was giving ridiculously high values (e.g. 350%).
Here is the raw data generated by the plate reader for a 1s read (Excel file) and a 0.1s (Excel file) read. Both reads have nearly identical values.
Isolated RNA from Abalone Dg samples (see below) using MoBio RNA PowerSoil Kit according to protocol. RNA was stored @ -80C.
Results: RNA quality looks great, as usual. Sample 06:6-44 has a very low yield, but was to be expected due to very small amount of starting tissue.
Inoculated 2 x 1L of 1x Marine Broth + biphenyl crystals (in 2L flasks) using 5mL of liquid culture from 20090501. Incubated at 28C 200RPM.
Transferred tissue from previously Chelexed samples:
WP3, WP7, WP13, WP14, WP15, CY1 – These all tested negative for virginica gDNA (18s qPCR by Rony on DATE)
CY42, CY36 – These tested positive for virginica gDNA (18s qPCR by Rony on DATE).
Used Qiagen DNeasy Kit. Digested samples for 3 hrs. at 55C in Proteinase K according to protocol.
Results: Horrible. Virtually no DNA in any of these samples. Additionally, the quality of the DNA is atrocious. Not sure what to do now.
Due to failure of gDNA isolation via the TriReagent method (see 20090511) used Qiagen DNeasy Kit. Digested samples for 3 hrs. at 55C in Proteinase K according to protocol. Performed on a subset of each site samples: BB#11-18 & DH#11-18.
Results: Excellent yields and superb quality.
Performed RT using Promega M-MLV RT according to M-MLV protocol and used 0.5ug oligo dT per ug of RNA on all BB and DH site samples that were negative for gDNA (see qPCR results 20090512). Calculations and work up are here. Samples were set up in a plate to facilitate sample loading in subsequent qPCRs.
UPDATE: cDNA plate was discarded 20120320 by SJW.
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
Performed qPCR on the DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the qPCR from 20090508 indicated residual gDNA was still present in some of the DNase treated RNA. Plate layout/set up is here.
Results: Samples BB# 2, 5, 6, 15 still came up as positive for gDNA contamination. These will NOT be used to make cDNA for subsequent qPCRs.
