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.

DNA Quantification – NcoI Linearized Withering Syndrome Plasmids from 20131224

Used Pico Green (Invitrogen) protocol for quantification on the Tecan plate reader. Used two standard curves for more accurate quantification; a “low” curve for the linearized plasmids and a “normal” curve for the complete plasmids. All samples and standards were done in replicates of two.

Low curve (ng): 0, 1, 5, 10, 20

Normal curve (ng): 0, 20, 40, 80

Results:

Low curve R2 = 0.99935

Normal curve R2 = 0.99738

Plasmids (ng/uL)

  • pCR2.1/WS1 – 32.613
  • pCR2.1/WS2 – 17.131
  • pCR2.1/WS3 – 72.836

NcoI Linearized Plasmids (ng/uL)

  • WS1 – 3.3956
  • WS2 – 1.5668
  • WS3 – 18.049

Will make dilutions of the linearized plasmids to prepare qPCR standard curves.

Plasmid Quantification – NcoI Linearized Withering Syndrome Clones from 20120726

Performed sample quantification on the NcoI linearized clones from 20120726. Used 1uL of sample in 100uL of a 1:200 pico green dilution and quantified using the Teacan plate reader.

Results:

Concentrations (ng/uL):

pWC8 – 12.616

p16RK3 – 12.909

p16RK7 – 2.6673

p18RK7 – 26.091

The standard curve is below. Raw fluorescence data, sample concentrations and plate layout is here.

y = 664.74x + 215.97

d = 212.4

r = 0.99974

Plasmid Curve Check – pCR2.1/AF133090

qPCR – Fresh RLP Plasmid Curve from earlier today

Ran qPCR on the new pCR2.1/AF133090 construct I created, in hopes of finally getting a working stand curve for the RLP qPCR assay. Master mix calcs are here. Additionally, three fecal extract DNA samples were run that Lisa was curious about quantifying (these have been run previously). All samples were run in duplicate. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

 

Plasmid Curve – Linearized pCR2.1/AF133090 from 20120430

Created a fresh dilution of this new construct using Low TE Buffer. Dilution calcs are here.

 

DNA Quantification – Linearized pCR2.1/AF133090 Construct from DATE

Performed DNA quantification using Pico Green and the Tecan plate reader, according to protocol.

Results:

The linearized construct was quantified as having 20.221ng/uL. Raw fluorescence, the mean quantity and plate layout are here. The results from the standard curve for quanting are below.

R2 = 0.99961

y = 503.74x + 221.84

d = 196.97

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 Extractions Comparisons – Test Water Filters

Trying out the following modifications to the filters to potentially improve extraction efficiency:

  1. 3rds (three tubes per filter; labeled as C0, C10, C100)

Each tube received twice the recommended volume of Buffer AL (400uL) and twice the recommend volume of Proteinase K. Samples were vortexed for ~30s and then incubated O/N @ 56C. Same samples were then combined into a single tube and passed through a Qiagen column. Samples were then processed according to the manufacturer’s protocol and eluted with 100uL of Buffer AE. Samples were then spec’d on the Teacan plate reader, along with the test water filter extractions from 20111212, using 100uL Pico Green solution (a 1:200 dilution of stock Pico Green in 1x Low TE Buffer) in each well and 1uL of template in each well.

Samples were then stored in Sam’s 4C Box.

Results:

Raw fluorescence readings, plate layout, and sample concentrations are here.

Standard Curve

y = 1807.1x + 2428.3

d = 98.525

r = 0.99997

Quick summary of results: No method appears to better than any of the others, as they all have very similar concentrations at each respective dilution. However, cutting the filter into halves is fastest when it comes to processing. Will qPCR to assess if there are noticeable differences between extraction methods at the molecular level.

DNA Extractions – Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here. Samples were quantified on 20111129 using 1uL of template, in 100uL of PicoGreen solution prepared in Low TE. After a few tries, a “low curve” was required to be used to accurately quantify the DNA in these samples.

Results:

y = 4010.4x + 4324.8

d = 115.35

r = 0.99997

Plate layout and average concentration of samples is here.

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