RNA from yesterday was treated according to Ambion Turbo DNA-free protocol to remove gDNA contamination. Work up is here.
Author Archives: kubu4
RNA Isolation – Abalone Dg Project Samples
RNA was isolated from the following samples using the MoBio RNA PowerSoil Kit according to protocol:
Results: RNA looks fabulous (as always from the PowerSoil kit). Will DNase treat the samples tomorrow.
DNase Treatment – Abalone Dg RNA isolated yesterday
RNA from yesterday was treated according to Ambion Turbo DNA-free protocol to remove gDNA contamination. Work up is here. Will check for residual gDNA contamination once I’ve finished with another set of RNA isolations from the remainder of Abalone Dg tissue.
PCR – C.pugetti DNA from 20090513 & 20090526
This is a repeat of yesterday’s PCR with a fresh working stock in hopes of eliminating the source of contamination seen in the negative control yesterday.
Lane 1 – 100bp ladder
Lane 2 – 5/13 DNA
Lane 3 – 5/26 DNA
Lane 4 – Empty
Lane 5 – H2O
Results: Same problem as yesterday. Water sample is contaminated. This suggests that the primer stocks are contaminated. Likely due to the use of non-sterile, “old” TE for reconstitution of the primer stocks. Will re-order them and reconstitute them sterily.
RNA Isolation – Abalone Dg Project
Isolated RNA using the MoBio RNA PowerSoil Kit according to protocol from the following samples:
06:6-45
06:6-49
06:6-50
06:6-51
06:6-52
08:4-6
08:4-9
08:4-10
Results: RNA looks fabulous (as always from the PowerSoil kit). Will DNase treat the samples tomorrow.
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.
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.
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.
DNA Methylation Test – Gigas site gDNA (BB & DH) from 20090515
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.
RNA Isolation – Abalone Dg Project samples
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.