Category Archives: qPCR Standard Curve

qPCR Standard Curve

qPCR – RLOv DNA Helicase Assay Limit of Detection

Continuing RLOv DNA Helicase qPCR assay validation.

This is the third of three plates to establish the assay’s limit of detection.

The first plate was run 20160121.

The second plate was run 20160122.

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.
  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

Plate layout, cycling params, etc can be seen in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2016-01-25 10-48-06_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-25 10-48-06_CC009827.pcrd

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qPCR – RLOv DNA Helicase Assay Limit of Detection

Continuing RLOv DNA Helicase qPCR assay validation.

This is the second of three plates to establish the assay’s limit of detection. The first plate was run yesterday (20160121).

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.

  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

Plate layout, cycling params, etc can be seen in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2016-01-22 10-15-55_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-22 10-15-55_CC009827.pcrd

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qPCR – RLOv DNA Helicase Assay Limit of Detection

Beginning RLOv DNA Helicase qPCR assay validation.

This is the first of three plates to establish the assay’s limit of detection.

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.

  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

Plate layout, cycling params, etc can be seen in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2016-01-21 15-12-31_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-21 15-12-31_CC009827.pcrd

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qPCR – RLOv DNA Helicase Standard Curve Check Repeat

Yesterday’s check of the RLOv DNA helicase standard curve using a freshly made 3 copy sample didn’t look perfect, so I’m re-running to see if things will tighten up a bit.

Repeated yesterday’s run with no changes.

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151223 – qPCR RLOv DNA Helicase Curve Check

Plate layout, cycling params, etc. can be found in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2015-12-24 11-02-59_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-24 11-02-59_CC009827.pcrd

 

Overall, things are looking better than yesterday’s run: better reps, better efficiency and better R^2. Will move forward with beginning to validate this qPCR assay, as well as use for some other sample analysis that Carolyn has in mind (comparing RLOv vs. WS levels in abalone collected from wild sites in California).

 

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qPCR – RLOv DNA Helicase Standard Curve Check

Since the standard curve for this assay was a bit wonky at the low copy number end the last time I ran it, I made a fresh 1:10 dilution of the 3 copies component of the curve (100μL of 30 copy sample in 900μL TE).

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151223 – qPCR RLOv DNA Helicase Curve Check

Plate layout, cycling params, etc. can be found in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2015-12-23 13-57-01_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-23 13-57-01_CC009827.pcrd

 

The efficiency & R^2 values look pretty solid, but the spacing between the 300 copy (Cq ~ 32 in the amplification plot) and the 30 copy (Cq ~ 34 in the amplification plot) samples is a bit too tight for my liking. Additionally, the reps for the 3 copy sample are very poor.

Will repeat to see if I can tighten things up…

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qPCR – pCR2.1/RLOv Standard Curves Testing

Earlier today, I created dilution series of the following two linearized plasmids to develop qPCR assays:

  • pCR2.1/RLOv_DNA_helicase
  • pCR2.1/RLOv_head_to_tail

Master mix calcs: 20151106 – qPCR RLOv Standard Curves

All samples were run in triplicate on a CFX96 thermal cycler (BioRad).

Cycling params, plate layout, etc. can be found in the qPCR Report (see Results below).

Results:
qPCR Data File (CFX96): Sam_2015-11-06 18-17-41_CC009827.pcrd
qPCR Report (PDF): Sam_2015-11-06 18-17-41_CC009827.pdf

DNA Helicase Curve

Amplifcation plots and the standard curve best fit line looks really good. Efficiency is very close to 100% and the R^2 = 0.99. Additionally, virtually all of the replicates are very tight. This looks like it will be totally usable as a standard curve for developing a qPCR assay that targets the RLOv DNA helicase gene.

 

Head-to-tail Curve

This curve is way wonky. Interestingly, the end-point fluroescence levels for this curve 5-fold lower than the DNA helicase curve. I’ll likely repeat this qPCR to see if these crappy results are repeatable. However, having a single qPCR assay (the DNA helicase standard curve) for RLOv detection/quantification might be sufficient, rendering a second qPCR assay unneeded.

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Standard Curve Dilutions – pCR2.1/RLOv Plasmids

Set up standard curve dilutions to use for qPCRs with the following two linearized plasmids:

  • pCR2.1/RLOv_DNA_helicase
  • pCR2.1/RLOv_head_to_tail

Used a spreadsheet developed by Nate many moons ago to determine necessary volumes based on plasmid size to obtain desired copy numbers.

Dilutions were made in TE Buffer.

DNA helicase dilutions (Google Sheet): 20151106 – Dilution table RLOv_DNA_helicase_qPCR_Standard_Curve

Head-to-tail dilutions (Google Sheet): 20151106 – Dilution table RLOv_head_to_tail_qPCR_Standard_Curve

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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.

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Restriction Digestions – pCR2.1/RLOv Plasmids

Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).

PLASMID Vol for 1.5μg (μL) H2O to 40μL
pCR2.1/RLOv_DNA_helicase 21.4 18.6
pCR2.1/RLOv_head_to_tail 11.1 28.9
pCR2.1/RLOv_membrane_gene_1 12.2 27.8
pCR2.1/RLOv_membrane_gene_2 14.3 25.7
pCR2.1/RLOv_tail_fiber 20 20

 

Digestion Master Mix

REAGENT SINGLE REACTION (μL) x 5.5 (μL)
Plasmid 40 NA
10x Buffer 3.1 (NEB) 5 27.5
BamHI (NEB) 1 5.5
H2O 4 22
TOTAL 50 Add 10μL to each tube

Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.

Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.

Results:

Hyperladder I (Bioline)

U = Undigested; Bam = BamHI digest

Besides the funky way this gel ran, the digests look to be complete.

Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).

 

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Sanger Sequencing Analysis – pCR2.1/RLOv Clones

Sequencing results from the samples that were submitted to Genewiz on Friday have come back:

  • SW01    RLOv_DNA_Helicase-M13F_-21_
  • SW02    RLOv_head_to_tail-M13F_-21_
  • SW03    RLOv_membrane_gene_1-M13F_-21_
  • SW04    RLOv_membrane_gene_2-M13F_-21_
  • SW05    RLOv_tail_fiber-M13F_-21_
  • SW06    RLOv_DNA_Helicase-M13R
  • SW07    RLOv_head_to_tail-M13R
  • SW08    RLOv_membrane_gene_1-M13R
  • SW09    RLOv_membrane_gene_2-M13R
  • SW10    RLOv_tail_fiber-M13R

The data (10-313205054_ab1.zip) has been stored in the following location: backupordie/sequencing_data/Sanger.

Sequences were loaded into Geneious (v.9.0.2). Vector sequences were trimmed/annotated using the Trim Ends with UniVec feature in Geneious.

Each clone was sequenced once from each direction, so the two sequences generated from each clone were mapped to the original sequence from which the primers were designed using Geneious Mapper.

The Geneious analysis was exported and saved in the following location:

backupordie/Sam/Sequencing_Analysis/Sanger/20151026_RLOv_clones_Sanger_analysis.geneious

Results:

Each clone’s sequence matches that of the source sequence, so we’re good to go!

Will proceed with dye-based quantification of each plasmid. Will then proceed with developing ISH probes (membrane genes 1 & 2, tail fiber gene) or qPCR standard curves (DNA helicase, head-to-tail).

In the alignments below, the reference sequence is highlighted in light yellow. The two electropherograms are align below the reference. The grey line in the consensus sequence indicates any sequence disagreements by placement of a black mark at the position. However, the sequences all match, so there are no black marks in the regions between the identified vector sequences (red annotations below each electropherogram).

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