Tag Archives: plasmid

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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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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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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Restriction Digestions – pCR2.1/RLOv Plasmids

Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).

PLASMID Vol for 1.5μg (μL) H2O to 40μL
pCR2.1/RLOv_DNA_helicase 21.4 18.6
pCR2.1/RLOv_head_to_tail 11.1 28.9
pCR2.1/RLOv_membrane_gene_1 12.2 27.8
pCR2.1/RLOv_membrane_gene_2 14.3 25.7
pCR2.1/RLOv_tail_fiber 20 20

 

Digestion Master Mix

REAGENT SINGLE REACTION (μL) x 5.5 (μL)
Plasmid 40 NA
10x Buffer 3.1 (NEB) 5 27.5
BamHI (NEB) 1 5.5
H2O 4 22
TOTAL 50 Add 10μL to each tube

Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.

Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.

Results:

Hyperladder I (Bioline)

U = Undigested; Bam = BamHI digest

Besides the funky way this gel ran, the digests look to be complete.

Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).

 

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