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.

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.

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

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

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

 

Plasmid Curve Check – pCR2.1/AF133090

qPCR – Fresh RLP Plasmid Curve from earlier today

Ran qPCR on the new pCR2.1/AF133090 construct I created, in hopes of finally getting a working stand curve for the RLP qPCR assay. Master mix calcs are here. Additionally, three fecal extract DNA samples were run that Lisa was curious about quantifying (these have been run previously). All samples were run in duplicate. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

 

Plasmid Curve – Linearized pCR2.1/AF133090 from 20120430

Created a fresh dilution of this new construct using Low TE Buffer. Dilution calcs are here.

 

DNA Quantification – Linearized pCR2.1/AF133090 Construct from DATE

Performed DNA quantification using Pico Green and the Tecan plate reader, according to protocol.

Results:

The linearized construct was quantified as having 20.221ng/uL. Raw fluorescence, the mean quantity and plate layout are here. The results from the standard curve for quanting are below.

R2 = 0.99961

y = 503.74x + 221.84

d = 196.97