qPCR – Abalone RLO qPCR assay

Still troubleshooting, as Lisa nor I, have had any luck to-date getting this back on track. Here I am testing out additional curves from 20120731 to determine if it is solely the p16RK7 curve (the one that we’ve used for all of the qPCR assay validation) is the problem. Master mix calcs are here.

Results:

qPCR Report (PDF): Sam_2013-10-01 10-51-45_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-10-01 10-51-45_CC009827.pcrd

 

PCR – Withering Syndrome cPCR Tests

Ran PCR using fresh working stock of primers.

Adjusted primer volumes being used. Recipe from Lisa (from the class) were only using 0.1uL of each primer per reaction.

Also, tried additional template DNA to determine if the template I’ve been using has become degraded. Additional template were two other WS plasmid curves from 20120731; 3e7. Master mix calcs are here.

All samples were run in duplicate.

Results:

Success!!!!! Unfortunately, I can’t say definitely if the previously used working stocks were bad or if the primer amount being used was too low (I suspect the latter), but it works and that’s all that matters. Will proceed with finding the limit of detection for this assay.

Chloroform Cleanup – EcoRI-digested Withering Syndrome Phage DNA from earlier today

To concentrate the sample, a chloroform cleanup and EtOH precipitation was performed. An equal volume of chloroform was added to the sample (220uL), vortexed for 30s and spun @ 16,000g at 4C for 15mins. The aqueous phase was transferred to a clean tube for EtOH precipitation.

0.1 volumes (16.8uL) of 3M NaOAC (sodium acetate; pH = 5.2) and 2.5 volumes (462uL) of ice cold 100% EtOH were added to the sample and mixed thoroughly. The sample was incubated O/N at -20C. The following day the sample was pelleted by spinning at 16,000g at 4C for 30mins. As expected, there was no visible pellet due to the extremely low quantity of DNA in the sample. The supernatant was discarded, the pellet was gently washed (no mixing/vortexing) with 70% EtOH, and spun @ 16,000g at 4C for 15mins. The supernatant was discarded the pellet was air dried for 15mins. The sample was reconstituted in 10uL of Buffer EB (Qiagen), which is simply 10mM Tris-HCl, and stored at 4C.

Gel Purification – EcoRI-digested pCR2.1 Vector from earlier today

Gel-excised band from earlier today was purified using the Ultra DA-free (Millipore) spin column according to the manufacturer’s protocol. Purified DNA was stored at 4C.

Restriction Digestions – Withering Syndrome Phage DNA and p16RK3

Performed restriction digestions on Phage RLO DNA (isolated on 20121130) and p16RK3 using EcoRI from NEB.

Phage RLO Digest

  • DNA – 192uL
  • 10x EcoRI Buffer – 22uL
  • EcoRI – 4uL
  • H2O – 2uL
  • TOTAL = 220uL

p16RK3 Digest

  • DNA (1ug) – 5.39uL
  • 10x EcoRI Buffer – 5uL
  • EcoRI – 1uL
  • H2O – 38.61uL
  • TOTAL = 50uL

Samples were incubated at 37C for 1hr. The p16RK3 sample was run on a 0.8% TBE gel to confirm digestion and isolate the vector-only band from the sample. The Phage RLO sample was subject to a chloroform cleanup and EtOH precipitation.

Results:

Digestion of Phage RLO DNA was not verified due to the extremely low quantities of DNA. In order to minimize loss of material, we will trust that the digestion was successful if the digestion of p6RK3 was successful.

Gel Loading

Lane 1 – Hyperladder I (Bioline)

Lane 2 – p16RK3 EcoRI

Neglected to run undigested p16RK3. However, the digestion pattern looks correct. The empty vector (pCR2.1; Invitrogen) should be 3931bp and we see the largest molecular weight band of the digest is running at ~4000bp. That band was excised and will be purified for subsequent ligation.

qPCR – Immomix Lot Number Comparison

Here we go again. Testing out Sammi’s vial of replacement batch lot #111A vs. my vial vs. lot #110C. Master mix calcs are here. Using the p16RK3 3e6 standard curve sample (from 20120730) as a positive control as well as a red abalone DNA fecal extract (extracted by Lisa 9/12/11; labelled “Red 1 +”) that Sammi used in her successful PCR with the lot #111A. Plate layout, cycling params, etc can be found in the Results (see below).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All three master mixes amplified AND look absolutely identical!!! How is this even possible? Whatever. I’ll go ahead and run the second of three plates to assess analytical sensitivity for the withering syndrome qPCR assay validation.

qPCR – Withering Syndrome qPCR Assay Validation: Analytical Sensitivity Plate # 1of 3

Performed analytical sensitivity assay validation using NcoI linearized p16RK7. Testing limit of detection by running 20 replicates each of 30 copies, 10 copies, 3 copies, 1 copy and 0 copies. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). This will be repeated two more times to have a final replicate count of 60 replicates of each copy number.

The positive control sample used was p16RK3 3e6 from 20120730.

Immomix Lot#IMX-110C

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

Results look really good. Detection all the way down to three copies and even some detection at one copy, however I’m not sure how that will hold up statistically.

The Cq values for the run are here. Baseline threshold was set to 400 RFUs and cycle # 41 in order to be compatible with previous qPCR data generated by Nate for this assay. Will get data entered into spreadsheet for statistical analysis.

qPCR – Withering Syndrome NcoI Linearized Clones from 20120726

Performed a dilution series to have the desired copy number using Low TE Buffer. The calculations were performed using a spreadsheet developed by Nate. The calculations are here and the workbook has a separate sheet for each clone. qPCR master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Reports below (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report – p16RK3 standards (PDF)

qPCR Report – p16RK7 standards (PDF)

qPCR Report – p18RK7 standards (PDF)

SUCCESS!!! It’s a friggin’ miracle! Finally, we have three clones (pWC8 did not amplify at all!) that are useable for the withering syndrome assay!! Here are the r^2 values and the efficiency for each of the three clones that did work:

p16RK3 – r^2 = 0.996, E = 91.7%

p16RK7 – r^2 = 0.995, E = 98.4%

p18RK7 – r^2 = 0.998, E = 94.1%

Due to it having the best efficiency, p16RK7 clone will be used for the assay validation.

UPDATED 20121024 – Modified data file (and subsequently the qPCR Report) to have a baseline threshold of 400 and cycles to analyze 41 to match existing conditions used for the withering syndrome qPCR assay validation. HOWEVER, I have NOT updated the r^2 and efficiency numbers listed above to reflect the new baseline setting! Please see the respective qPCR Reports for that information!!

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

Restriction Digests – Withering Syndrome Clone Plasmids from 20120718

Performed restriction digest on all four clones using NcoI. Reactions were run for 1hr @ 37C and then heat inactivated @ 65C for 20mins.

Each reaction contained:

Plasmid – 5uL

10x Buffer – 5uL

NcoI – 1uL

H2O – 39uL

After inactivation, 5uL from each reaction were run on a 1% TBE gel to confirm digestion. 1uL of undigested plasmid was run along side the corresponding digest.

The four clones are referred to as:

  • pWC8
  • p16RK3
  • p16RK7
  • p18RK7

Results:

Gel Loading (left to right):

Lane 1 – Hyperladder I (Bioline)

Lane 2 – pWC8 (Und.)

Lane 3 – pWC8 (NcoI)

Lane 4 – p16RK3 (Und.)

Lane 5 – p16RK3 (NcoI)

Lane 6 – p16RK7 (Und.)

Lane 7 – p16RK7 (NcoI

Lane 8 – p18RK7 (Und.)

Lane 9 – p18RK7 (NcoI)

Lane 10 – Hyperladder I (Bioline)

All digests are complete. All clones reveal the same restriction digestion pattern, producing two bands: ~2500bp and ~3000bp. The band sizes total ~5500bp, which is in line (5432bp) with the full withering syndrome 16s clone in the pCR2.1 TOPO vector (Invitrogen). Will quant and prepare a dilution series for qPCR.

Plasmid Isolation – Withering Syndrome Cultures from Yesterday

Isolated plasmid DNA from 3mL of each culture inoculated yesterday using QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer’s protocol. Eluted the plasmid DNA with 50uL of Buffer EB and spec’d on Roberts Lab NanoDrop1000.

Results:

Samples look fine; good yields, good OD260/280 ratios. Will perform NcoI restriction digests on samples to linearize them in preparation for use in a new standard curve.

Bacterial Cultures – Withering Syndrome Clones

Inoculated 5mL of LB + Amp (100ug/mL) in 50mL conicals with one of the following Withering Syndrome clones frozen stocks (located in -80C box called “WS RLP Plasmids”):

  • PWC8 WFS-RLP pCRII (from 4/13/01)
  • p16RK3 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
  • p16RK7 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
  • p18RK7 WFS-RLP rDNA pCR2.1 (from 4/15/01)

Incubated O/N, 37C, 200RPM. Will isolate plasmid DNA tomorrow.