Tag Archives: qPCR standard curve

qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs were re-used from previous standard curve check (Google Sheet): 20161128 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2017-07-03 09-34-31_CC009827.pdf
qPCR Data File (CFX): Sam_2017-07-03 09-34-31_CC009827.pcrd

Curve looks good! Will use this one going forward. Will store in the withering syndrome standard curve box in the FSH 240 4C.

 

ALL MIXES

 

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Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

The last dilution I made was jacked up, so I made a new one today.

Used the 3e7 dilution of p18RK7 from 20161128 as the “stock” to make serial, 1:10 dilutions (final volume of 1000uL in each tube).

I prepared the dilution series using lab-made Low TE Buffer (pH=8.0). The original dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table

Will test out the new curve dilution shortly.

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qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs were re-used from previous standard curve check (Google Sheet): 20161128 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2017-06-29 12-49-46_CC009827.pdf
qPCR Data File (CFX): Sam_2017-06-29 12-49-46_CC009827.pcrd

 

I screwed something up in the dilution series. Seems difficult to screw up something so basic, but the results don’t lie. Will discard this curve and will remake the curve and re-test. Argh!

 

 

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Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

The withering syndrome standard curve on my last two qPCRs has been wonky, so I’m making a new curve.

Used the 3e7 dilution of p18RK7 from 20161128 as the “stock” to make serial, 1:10 dilutions (final volume of 1000uL in each tube).

I prepared the dilution series using IDTE (pH=8.0; IDT) Buffer. The original dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table

Will test out the new curve dilution shortly.

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qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs (Google Sheet): 20161128 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2016-11-28 11-02-46_CC009827.pdf
qPCR Data File (CFX): Sam_2016-11-28 11-02-46_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve to past versions of the curve. Will put new curve in the standard WS plasmid curve box in FSH240 fridge.

 

ALL MASTER MIXES

 

 

 

MASTER MIX #1


 

MASTER MIX #2

 

 


MASTER MIX #3

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Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

Lisa recently ran a qPCR and noticed that the standard curve had drifted quite a bit and was no longer usable, so I’m making more.

Used NcoI-linearized p18RK7 abalone withering syndrome plasmid (from 20120726) to make a fresh dilution series. The plasmid was most recently re-assessed and successfully used for a new standard curve on 20160316. As such, I re-used the dilution calculations from 20160316 (see sheet below).

I prepared the dilution series using IDTE (pH=8.0; IDT) Buffer. The dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table

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qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs (Google Sheet): 20150316 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2016-03-16 17-04-05_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-16 17-04-05_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve. Will use this for future withering syndrome qPCR assays and will send an aliquote of each dilution to Colleen Burge.

MASTER MIX #1


 

MASTER MIX #2

 


MASTER MIX #3

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Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

We are virtually out of the withering syndrome qPCR standard curve plasmid dilutions and I need to send some to Colleen Burge at Univ. of Maryland Baltimore County. As such, I’ll need to make a fresh dilution series and test it out prior to sending to her.

Used NcoI-linearized p18RK7 abalone withering syndrome plasmid (from 20120726) to make a fresh dilution series.

Since it’s been nearly 4yrs since the plasmid was prepared, I decided to re-quantify the DNA using a Qubit3 (Life Technologies) in the Roberts Lab. Used the dsDNA BR Qubit reagents.

SAMPLE CONCENTRATION (ng/μL)
p18RK7 NcoI-linearized 29.0

The result is pretty close to what had been measured 4yrs ago (~26.1ng/μL – difference of  ~10%), so that’s good to see.

I prepared the dilution series using IDTE (pH=8.0; IDT) Buffer. The dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table

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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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