qPCR – Withering Syndrome ddPCR Comparison

Performed qPCR on the same exact samples previously sent to Alice Nguyen at the Marine Science Institute for digital droplet PCR (ddPCR) to compare our results to hers. These samples were previously quantified as part of the Ab Endo Project (Ab Endo 2011 Water Filter DNA samples). Here’s the list of samples that were sent to (and back from) Alice:

  1. CI SRI CP 1A
  2. CARMEL +500M 2
  3. CI SRI CP 2B
  4. CI SRI CP 2A
  5. CI SRI CP 1B
  6. CARMEL +500M 1

Master mix calcs are here: 20150415 – qPCR WS Ab Endo vs ddPCR

Standard curve was p16RK7 (from 20120730).

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

Results:
qPCR Report (PDF): Sam_2015-04-15 15-38-13_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-04-15 15-38-13_CC009827.pcrd

Table – Summary of qPCRs and ddPCR data

Sample Number Sample Name Initial qPCR Copies ddPCR Copies Today qPCR Copies
AB01 CI SRI CP 1A 0  3.8  0
AB02 CARMEL +500M 2 1.57  8.7  1.3
AB03 CI SRI CP 2B 147  104  59.2
AB04 CI SRI CP 2A 0  175  83.1
AB05 CI SRI CP 1B 74.8  2.8  0
AB06 CARMEL +500M 1 1.08  4.9  0

The table certainly shows some inconsistency, both between ddPCR and qPCR. Additionally, there is also inconsistency between the initial qPCRs and today’s qPCRs. Due to the limited sample volumes remaining for these sub-samples that were sent to Alice and then returned to us, I will repeat the qPCR using the stock DNA and see how that compares.

qPCR – New Withering Syndrome Plasmid Curve (from 20131106)

Ran qPCR on new p16RK7 plasmid curve.

Master mix calcs are here: 20131119 – qPCR RLO New Curves

Results:

qPCR Report (PDF): Sam_2013-11-19 11-56-58_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-11-19 11-56-58_CC009827.pcrd

No amplification of either curve! This sucks. Will brainstorm additional troubleshooting ideas…

Plasmid Curve Dilution – Withering Syndrome Linearized Plasmid (p16RK7) from 20131114

Prepared dilutions of linearized p16RK7 for use as new qPCR standard curve for the withering syndrome qPCR assay.

Calculations were performed using a spreadsheet set up by Nate.

Calculations are here: 20131118 – Dilution table for creating QPCR plasmid standard curve

 

Restriction Digest – Withering Syndrome Clone Plasmid (p16RK7) from 20131106

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

Each reaction contained:

  • Plasmid – 5uL
  • 10x NEB Buffer 3 – 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 digest.

Results:

Plasmid Isolation – p16RK7 A Withering Syndrome 16s Clone for Fresh Plasmid Curve

Due to the recent failure of the plasmid standard curve for the withering syndrome qPCR assay, we are creating a fresh plasmid prep. Inoculated 5mL of LB+100ug/mL ampicillin from a frozen stock of p16RK7 C19. Incubated O/N @ 37C on a rocker. Isolated plasmid DNA with Qiagen Mini Prep Spin Kit, using 3mL of culture, according to manufacturer’s protocol.

qPCR – Abalone RLO qPCR Assay

Testing out new stocks of primers and probe received this week. Stocks (and working stocks) will be made with different batches of low TE and PCR water than what we have been using in the hood. Will use the RLO p16RK7 plasmid curve from 20120731. Master mix calcs are here.

Results:

qPCR Report (PDF): Sam_2013-10-09 13-33-42_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-10-09 13-33-42_CC009827.pcrd

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

 

qPCR – Abalone RLO qPCR assay; trying “new” reagent aliquots and additional plasmid curve

Since yesterday’s comparison of thermal cyclers yielded identical results, it’s clear that the machine is not the issue. I’m repeating yesterday’s qPCR, but using the aliquots of reagents from Lisa that I used on DATE. Additionally, I’m checking a fresh, separate dilution of the RLO plasmid curve (p16RK7 from 20120731) that Lisa made last week. Master mix is the same as yesterday.

Results:

qPCR Report (PDF): Sam_2013-09-25 10-52-18_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-09-25 10-52-18_CC009827.pcrd

qPCR – Comparing CFX96 (Friedman Lab) vs Opticon 2 (Roberts Lab) for abalone RLO qPCR assay

Due to recent problems with the RLO qPCR assay experienced by both Lisa and me, I am going to run the same master mix on both the machines mentioned above to evaluate whether the Friedman Lab CFX96 is the cause of our recent problems with this assay.

Ran the RLO plasmid curve (p16RK7 from 20120731), each sample in duplicate. Master mix calcs are here.

Results:

qPCR Report (CFX96; PDF): Sam_2013-09-24 13-51-54_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-09-24 13-51-54_CC009827.pcrd

qPCR Data File (Opticon 2)

 

Opticon 2 Amplification:

CFX96 Amplification:

qPCR – Testing RLO Plasmid Curve with Lisa’s Working Stocks

Lisa has recently been running the abalone RLO qPCR assay and it her standard curve has looked fine. Set up with her working stocks of reagents of Lisa’s. Ran the RLO plasmid standard curve (p16RK7 from 20120731). Master mix calcs are here.

Results:

qPCR Report (PDF): Sam_2013-09-11 14-27-40_CC009827.pdf
qPCR Data File (CFX96): Sam_2013-09-11 14-27-40_CC009827.pcrd