Plasmid Isolation – Withering Syndrome Plasmids

Selected colonies 1, 2 & 3 from the colony screens from 20131205 for plasmid isolation. 5mL of liquid cultures were grown O/N at 37C. 1mL of each culture was mixed with 1mL of sterile 50% glycerol solution and stored @ -80C. 3mL of each culture was used for plasmid isolation. Plasmids were isolated using the Qiaprep Mini Spin Kit (Qiagen) according to protocol. Plasmids were eluted with 50uL of Buffer EB.

qPCR – Withering Syndrome Assay with New IDT Primers

It dawned on me that the qPCRs I ran on 20140909 comparing different stocks of primers, using the new IDT probe (which all ended up looking bad), all used primer stocks that had been reconstituted with low TE buffer made in the lab, as opposed to “store bought.” Could this actually be the reason that the IDT probes (which come lyophilized and require reconstitution prior to use) don’t seem to work, while the ABI probe (which comes as a liquid, 100uM stock) does?

Received new IDT primer stocks of WSN1F/R and reconstituted them with the store bought low TE buffer from IDT.

All samples were run in duplicate. See the qPCR Report (in Results below) for plate layout, cycling params, etc.

Baseline Threshold set to 580 RFUs, as previously determined by Lisa for use with the Promega master mix.

Results:

qPCR Report (PDF): Sam_2014-09-19 10-55-33_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-09-19 10-55-33_CC009827.pcrd

I’ll be damned! Our lab-made low TE buffer seems to have been the culprit! Both IDT and ABI probes performed perfectly and are virtually identical. See the amplification plots and standard curve plots below for each of the two probes. Both exhibit R^2 values of 0.999.

ABI Probe Amplificaton

ABI Probe Standard Curve

IDT Probe Amplification

IDT Probe Standard Curve

qPCR – Withering Syndrome Probes Comparison

Due to poor performance seen in yesterday’s second run, which used the IDT probe instead of the ABI probe, I will run the p18RK7 curve (from 20120731) with both the IDT and the ABI RLP_p probes to see if the probe is the underlying issue.

Master mix calcs are here: 20140827 – qPCR IDT-ABI Probe Comparison

All samples were run in duplicate.

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

Baseline threshold set to 580 RFUs.

Results:

qPCR Report (PDF): Sam_2014-08-27 10-32-19_CC009827.pdf

qPCR Date File (CFX96): Sam_2014-08-27 10-32-19_CC009827.pcrd

Quick summary: IDT probe is bad, ABI probe is good.

However, I’ve just realized that there might be more to this than just a bad probe from IDT. The amplification profile is suspiciously similar to what we were seeing with the bad probe(s) from Biosearch Technologies. The only thing that differentiates the IDT and Biosearch Technologies probes from the ABI probe is that the ABI probe already arrived reconstituted at 100uM. Biosearch Technologies and IDT probes arrive lyophilized and have to be reconstituted by us. Is it possible that the buffer we’re using is degrading the probes? Will order new IDT probe and order IDT’s version of low TE buffer (called IDTE).

Here are the amplification and standard curve plots:

IDT Amplification

IDT Standard Curve

ABI Amplification

ABI Standard Curve

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Ran limit of detection qPCR for the Promega 2x GoTaq Probe Master Mix using p18RK7 (from 20120730) primary curve and p18RK7 low curve (from 20140507).

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

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

Results:

qPCR Report (PDF): Sam_2014-05-21 14-04-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-21 14-04-07_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Ran limit of detection qPCR for the Promega 2x GoTaq Probe Master Mix using p18RK7 (from 20120731) primary curve and p18RK7 low curve (from 20140507).

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

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

Results:

qPCR Report (PDF): Sam_2014-05-21 10-07-34_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-21 10-07-34_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection

NOTE: Well B6 is an outlier. Without that sample, the R^2 changes from 0.977 to 0.990. The Cq value for this sample has been highlighted in its entry on the Promega Master Mix Withering Syndrome Limit of Detection spreadsheet.

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Ran limit of detection qPCR for the Promega 2x GoTaq Probe Master Mix using p18RK7 (from 20120731) primary curve and p18RK7 low curve (from 20140507).

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

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

Results:

qPCR Report (PDF): Sam_2014-05-20 13-17-13_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-20 13-17-13_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection

qPCR – p18RK7 Low Curve Check

I needed additional dilutions for a low curve to finish validating the Promega 2x GoTaq Probe Master Mix limit of detection. Will test the low curve before continuing the full limit of detection process.

Master mix calcs are here: 20140507 – qPCR p18RK7 low curve check

Results:

qPCR Report (PDF): Sam_2014-05-07 14-47-55_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-07 14-47-55_CC009827.pcrd

New low curve looks good. Will use this for subsequent limit of detection tests.

qPCR – Promega 2x GoTaq Probe Master Mix Withering Syndrome Assay Limit of Detection

Now that we’ve decided to go with the Promega master mix, we need to re-do the limit of detection to validate the assay, as far as sensitivity goes.

Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1

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

Results:

qPCR Report (PDF): Sam_2014-05-06 14-38-39_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-06 14-38-39_CC009827.pcrd

Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection