DNA Quantification – BamHI-Linearized pCR2.1/RLOv plasmids

Quantified the linearized RLOv plasmids using the Quant-It DNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards (10μL each of 0, 5, 10, 20, & 40ng) were run in triplicate.

Linearized plasmids were quantified in replicates of six.

Quantification was performed in black 96-well plates in the Seeb Lab Victor3 1420 (Perkin Elmer) plate reader. See the spreadsheet linked in the Results below for reading protocol.

Results:

Calculations & raw fluorescence readings (Google Sheet): 20151106_quantification_RLOv_linearized_plasmids

Standard Cuve R^2 = 0.999

Best Fit Equation: y = 1174.8215x + 8919.308333333

PLASMID CONCENTRATION (ng/μL)
pCR2.1/RLOv_DNA_helicase 15.498
pCR2.1/RLOv_head_to_tail 17.887
pCR2.1/RLOv_membrane_gene_1 25.111
pCR2.1/RLOv_membrane_gene_2 28.264
pCR2.1/RLOv_tail_fiber 23.442

Will proceed to making qPCR standard curves from the DNA helicase and the head-to-tail linearized plasmids.

Plate Reader – Tecan Plate Reader Problem

The Tecan plate reader is behaving strangely. I tried to quantify plasmid DNA samples isolated on Friday, but this is how the plate turned out:

The areas NOT demarcated by the black lines should all appear as dark blue (i.e. no fluorescence detected). I’ve never seen a plate look like this before.

To be sure that the issue wasn’t specific to just the plate I was using, I ran an empty plate through the same Tecan method.

Here’s the pic of an entirely empty/blank plate (i.e. no samples loaded in any wells; just an empty, dry plate):

Empty Plate

Ideally, these should all be the same exact color (dark blue), since there should be no fluorescence in any wells.

I probably need to contact Tecan regarding this issue…

DNA Quanting – Black Abalone Digestive Gland DNA

Quantified gDNA from the following samples in preparation for high-throughput sequencing by Stan Langevin’s group. Quantification was done using Pico Green, Tecan plate reader and Magellan 6 software. The r^2 value of the standard curve was 0.9986 and replicates showed little variation.

Concentrations below are the mean of three replicates and are in ng/uL.

  • 06:5-28 – 42.4
  • 06:6-41 – 60.3
  • 06:6-44 – 57.1
  • 06:6-53 – 72.1
  • 06:6-54 – 40.1
  • 06:6-55 – 40.5
  • 06:6-66 – 40.6

Stan Langevin has requested at least 50ng of DNA from each sample. I will aliquot ~100ng of each sample into individual tubes. He will be picking up aliquots tomorrow morning for sequencing on the MiSeq (Illumina).

DNA Quantification – Digested RLP Plasmid from 20120412

Quanted DNA with PicoGreen on the Teacan plate reader.

Results:

R2 = 0.99867

y = 746.42x + 2487.7

Overall, curve looks pretty good.

Raw fluorescence and plate layout is here.

Created a fresh RLP plasmid curve using the NcoI-digested p16RK3-A plasmid prep from 20120412. Standard curve calcs were performed using Nate’s spreadsheet and is linked here. Curve was made using Low TE Buffer and will be stored @ 4C.

Plasmid Curve – RLP Standard Plasmid Curve

DNA Quantification – NcoI Linearized RLP Plasmid DNA from Yesterday

Performed DNA quantification according to Friedman Lab protocol using Pico Green and the Teacan plate reader.

Results:

R2 = 0.99989

y = 662.22x + 624.42

Plate results (mean concentration in ng/uL) are here. Standards on the plate used were 0, 1, 5, 10, 20 ng/uL.

Quanting standard curve looks spectacular.

NcoI linearized RLP plasmid = 18.21 ng/uL

Will make a fresh RLP plasmid curve and check with qPCR.

Upon attempting to prepare the dilutions using one of Nate’s dilution calculation sheets, I realized that the dilution sheets that I looked at were inconsistent in regards to the size of the linerarized RLP plasmid! The sheet that Lisa most recently used (2/29/2012) indicates that the RLP plasmid is 5454bp in size! This is definitely incorrect as can be seen by my gel from yesterday (the linearized fragment doesn’t run greater than 5000bp. Additionally, Nate has other calculation sheets that indicate the plasmid size is 4133bp! This seems to jive both with my gel results from yesterday. However, this suggests that the last curve that Lisa made was incorrect because the calculations were based off of an incorrect plasmid size.

This discrepancy could explain the shift in the most recent curves to coming up at later Cqs than what Nate’s old curve had been coming up at, since using the plasmid size of 5454bp would assume you had more copies of plasmid than were actually there. Thus, the calculated copy number would actually be less than expected at each dilution point of the plasmid curve.

Will need to discuss with Lisa to find out exact size of plasmid with RLP insert.

20120406 – UPDATE: Carolyn has just remembered that there are multiple, different versions of RLP cloned into a variety of vectors. After discussion with Carolyn and Robyn, Carolyn definitively determined that Lisa and I had not been using the correct clones with the correct RLP insert! The correct vector/insert should be p16RK3-C with the entire AF133090 sequence insert.

DNA Quantification – Volume Comparison and Filter Extracts (from 20111107) Comparison

Compared effects of well volume for standards, and measured filter extracts from (20111107 and 20111108). For the volume comparison, 1uL of standards were placed in 200uL or 100uL of diluted PicoGreen (1:200). All filter extracts were measured using 1uL of sample in 100uL of diluted PicoGreen (1:200). Plate layout and raw fluorescence readings are here.

Results:

DNA Quantification – Dye Comparison

After learning that Lisa recently had issues quantifying some samples, I decided to troubleshoot the quantification protocol. In that troubleshooting, I discovered that the dye in the Quant-IT Kit (Invitrogen) fluoresces outside of the excitation/emission wavelengths set up on the plate reader (Tecan). It turns out that previous kits being used were with Pico Green (Invitrogen), which has the “appropriate” excitation/emission wavelengths that the plate reader is set up to use!

So, I did a dye comparison as well as a template volume comparison.

Results:

Briefly, the Pico Green dye works better than the dsDNA BR dye, which is not surprising considering that the Pico Green fluoresces in the wavelengths that the plate reader is set up to detect! Additionally, we see a “swamping” of the system when using the 100ng/uL standard when we use a volume of more than 1uL.

As such, I used the set of samples containing 1uL of template as the standard curve for the plate (see below).

y = 222.75x + 413.69

d = 255.77

r = 0.99954

DNA Quantification – Abalone Water Filters Extraction Comparisons

Quantified DNA extracted from water sample filters. Samples were extracted by Lisa. Thus, I’m unsure what those samples were and if they were from the same filter or not. Samples were extracted using the following methods:

MoBio Kit (MB)

DNeasy Kit (DNE)

1 Step extraction (1 STEP)

NOAA extraction (NOAA)

Samples were quantified using the Tecan plate reader (w/Magellan 6 software) and the Quant-IT Kit (Invitrogen) according to the Friedman Lab protocol.

Results:

The standard curve exhibited too much drift between dilutions. For example, the difference in fluorescence between the 100ng standard and the 10ng is only ~6.4-fold. It should be a 10-fold difference. This is surprising, since these standards are factory produced (i.e. not a dilution created by me).

Additionally, the best fit line equation from the standard curve results in a negative concentration for the 1ng standard. As all four samples evaluated in this run exhibit fluorescence below that of 1ng, an accurate concentration cannot be calculated based on this standard curve equation.

Consulted Carolyn regarding the curve results. She has suggested that I re-do extractions using a single filter split into four and then re-do the quanting with a more detailed low end. Will also add more replicates to all standards and samples run.

Standard Curve

Best fit line info is:

y=344.52x + 2773

r squared = 0.99379

Raw fluorescence readings (top half) and plate layout (bottom half). Excel file