---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2012-03-27 00:15:02+00:00
layout: post
slug: qpcr-taylor-water-filter-dna-extracts-from-20120322
title: qPCR - Taylor Water Filter DNA Extracts from 20120322
  - Sam White
categories:
  - 2012
  - Miscellaneous
tags:
  - CFX96
  - DNA
  - qPCR
  - Vibrio tubiashii
  - VtpA
  - water filter
---

Ran qPCR on the Taylor water filter DNA extracts from [20120322](/Sam%27s+Working+Notebook+January+-+March+2012#sjw20120322) using V.tubiashii VtpA primers (provide by Elene; no SR ID?) instead of 16s primers, which failed to produce acceptable results in the melt curves (see [20120323](/Sam%27s+Working+Notebook+January+-+March+2012#sjw20120323)). Additionally, Elene has a standard curve for V. tubiashii (from 1/12/2011) based off of CFUs/mL, which will allow us to quantify theoretical number of V.tubiashii CFUs present in each sample.

[Master mix calcs are here](https://eagle.fish.washington.edu/Arabidopsis//Notebook%20Workup%20Files/20120326-01.jpg). Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

[qPCR Date File](https://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-03-26%2012-54-03_CC009827.pcrd) (CFX96)

[qPCR Report](https://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-03-26%2012-54-03_CC009827.pdf) (PDF)

Overall, the run looks excellent. Both negative controls and no template controls are clean. Since I was able to use a standard curve, I determined CFUs of V.tubiashii in each sample, as follows:

Mean CFUs per qPCR reaction / template volume per qPCR reaction x filter extraction elution volume (100uL) = total CFUs on water filter.

Total CFUs on filter / filtered water volume = **CFUs per mL in Taylor tanks**

**158** - 16500 copies/2uL = 8250 copies/uL x 100uL = 825000 copies on water filter/1000mL = **825 copies/mL**

**200** - 5700 copies/2uL = 2850 copies/uL x 100uL = 285000 copies on water filter/1000mL = **285 copies/mL**

**279** - 325000 copies/2uL = 162500 copies/uL x 100uL = 16250000 copies on water filter/1000mL = **16250 copies/mL**

**313** - 152 copies/2uL = 76 copies/uL x 100uL = 7600 copies on water filter/1000mL = **7.6 copies/mL**

**341** - 124000/2uL = 62000 copies/uL x 100uL = 6200000 copies on water filter/1000mL = **6200 copies/mL**

**410** - 132000/2uL = 66000 copies/uL x 100uL = 6600000 copies on water filter/1000mL = **6600 copies/mL**

**433** - 63700/2uL = 31850 copies/uL x 100uL = 3185000 copies on water filter/1000mL = **3185 copies/mL**

**503** - 110/2uL = 55 copies/uL x 100uL = 5500 copies on water filter/1000mL = **5.5 copies/mL**

**551** - 2000/2uL = 1000 copies/uL x 100uL = 100000 copies on water filter/1000mL = **100 copies/mL**

**604** - 272/2uL = 136 copies/uL x 100uL = 13600 copies on water filter/1000mL = **13.6 copies/mL**

Sample #410 was from the only tank that exhibited mortalities and was the only group of oyster larvae that showed any expression from the V.tubiashii genes (see DATE).