Jul 31, 2020
16S Bacteria 338F-516P-805R BSA
Forked from a private protocol
- 1Soil and Water Research Infrastructure
Protocol Citation: Roey Angel, Eva Petrova, Ana Lara 2020. 16S Bacteria 338F-516P-805R BSA. protocols.io https://dx.doi.org/10.17504/protocols.io.m7qc9mw
Manuscript citation:
Yu, Y, Lee, C, Kim, J & Hwang, S (2005) Group-specific primer and probe sets to detect meth-anogenic communities using quantitative real-time polymerase chain reaction.Biotechnology and Bioengineering 89:670–679. url:http://dx.doi.org/10.1002/bit.20347
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 12, 2018
Last Modified: September 01, 2020
Protocol Integer ID: 10192
Abstract
Universal 16S rRNA BActeria 338F-516P-805R
BAC338F ACT CCT ACG GGA GGC AG , target E.coli : 338-354, Yu et al. (2005), B&B
BAC516P* TGC CAG CAG CCG CGG TAA TA, target E.coli : 516-536, '
BAC805R GAC TAC CAG GGT ATC TAA TC , target E.coli : 785-805, '
* Probe must be dual-labelled either with 5’-6-FAM, 3’-BHQ1 or any other valid combination.
Materials
MATERIALS
iQ™ SYBR® Green SupermixBio-Rad LaboratoriesCatalog #1708880
QPCR mixture
QPCR mixture
| Reagent | Final conc. | 1 tube (20μl) | plate (20μl x 100) | |
| PCR H2O | 4.6 | 460 | ||
| iQTM Supermix | 1x | 10 | 1000 | |
| MgCl2 (25mM) | 4.0 mM | 0.8* | 80* | |
| BSA (20μg/μl) | 0.2 μg/μl | 0.2 | 20 | |
| 338F (10μM) | 0.5 μM | 1.0 | 100 | |
| 805R (10μM) | 0.5 μM | 1.0 | 100 | |
| 516P (10μM)† | 0.2 μM | 0.4 | 40 | |
| Template | 2 | 2 x 100 |
* Buffer contains MgCl2 at final conc. of 3.0 mM
Program
Program
1. 95◦C – 5′
2. x 40 {
a. 95◦C – 30′′
b. 22◦C – 60′′ take a snapshot
}