Tag Archives: M1

qPCR – Test Australian OsHV-1 ORF117 Primers

Using primers I previously designed, I tested them out for functionality (using the clone #1 plasmid prep DNA I made previously) and specificity (using the Australian, California, & French variants recently received)

Created a working 1:100 dilution of ALL DNA tested here.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20171221 – qPCR Austrailian OsHV-1 ORF117 Primer Test

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

Results:
qPCR Report (PDF): Sam_2017-12-21 15-09-49_CC009827.pdf
qPCR Data File (CFX): Sam_2017-12-21 15-09-49_CC009827.pcrd

Firstly, the primers work and generate a single melt curve peak (see melt curve plot below); so that proves functionality.

Results are interesting.

Australian samples (plasmid and DNA) amplify.

French samples (M1 & M2) do not amplify.

California samples: 3 of 4 samples amplify.

It’s possible that the California sample that did not amplify is due to too little DNA present in the 1:100 dilution I used (or, possibly no DNA is present at all). I have not quantified the DNA in these samples – went off assumption that the samples had previously been confirmed to have DNA in them by the source laboratories.

Regardless, the primers used here will amplify the French variant, but will amplify Australian and Californian variants.

See labeled amplification plots below.

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Agarose Gel – Oly gDNA for BS-seq Libraries, Take Two

The gel I ran earlier today looked real rough, due to the fact that I didn’t bother to equalize loading quantities of samples (I just loaded 1μL of all samples regardless of concentration). So, I’m repeating it using 100ng of DNA from all samples.

Additionally, this gel also includes C.gigas samples that Katie Lotterhos sent to us to see how they look.

Ran a 0.8% agarose, low-TAE gel, stained with ethidium bromide.

Results:

 

Look at that! The samples look MUCH nicer when they’re not overloaded and uniformly loaded!

Most have a prominent high molecular weight band (the band that’s closes to the top of the ladder, not the DNA visible in the wells). All exhibit smearing, but 2NF1 shows a weird accumulation of low molecular weight DNA.

Katie’s C.gigas samples (M1, M2, M3) look similar to the Olympia oyster gDNA, however her samples appear to have residual RNA in them (the fuzzy band ~500bp).

Will discuss with Steven which samples he wants to use for bisulfite treament and library construction.

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