Jan 04, 2016
16s Pyrotag PCR
- Howard Ochman1
- 1Matthew Sullivan Lab, University of Arizona/Ohio State University
- VERVE Net
- Sullivan Lab
Protocol Citation: Howard Ochman 2016. 16s Pyrotag PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.c5iy4d
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
Created: June 19, 2015
Last Modified: February 07, 2018
Protocol Integer ID: 906
Keywords: pyrotag pcr, pcr, engelbrekson ochman
Abstract
Modified from H. Ochman protocol: Environ. Micro. 2009 Kunin, Engelbrekson Ochman & Hugenholtz
Guidelines
Protocol Using Takara Hot Start EX Taq (cat. no RR006A)
| Pyrotag PCR Master Mix | ||
| Reagent | Volume μl | |
| Water | 18.25 | |
| 10x EX Buffer II | 2.5 | |
| dNTP mix (2.5mM ea) | 2 | |
| F primer (10 μM) | 0.5 | |
| R primer (10 μM) | 0.5 | |
| Taq (5U/ μl) | 0.25 | |
| DNA template | 1 |
| Cycling Conditions | |||
| Temp. °C | Time (min) | Cycles | |
| 95 | 3:00 | ||
| 95 | :30 | 25 | |
| 55 | :45 | ||
| 72 | 1:30 | ||
| 72 | 10:00 | ||
| 15 | Hold |
Protocol Using Sigma Taq (cat. no. D4545) and dNTP mix (cat. no. D7295)
| Pyrotag PCR Master Mix | ||
| Reagent | Volume μl | |
| Water | 17.75 | |
| 10X Buffer w/o MgCl2 | 2.5 | |
| MgCl2 (25mM) | 2 | |
| dNTP mix (10mM ea) | 0.5 | |
| F primer (10 μM) | 0.5 | |
| R primer (10 μM) | 0.5 | |
| Taq (5U/μl) | 0.25 | |
| DNA template | 1 |
| Cycling Conditions | |||
| Temp. °C | Time (min) | Cycles | |
| 95 | 3:00 | ||
| 95 | :30 | 25 | |
| 55 | :45 | ||
| 72 | 1:30 | ||
| 72 | 10:00 | ||
| 15 | Hold |
Reconditioning PCR sample (from Nucleic Acids Research 2002 Thompson, Marcelino & Polz): Make a 1:10 dilution of PCR product into fresh PCR mix; cycle at 95°C for 3 minutes, then 3 cycles of 95°C for 30 second, 55°C for 45 seconds, and 72°C for 1.30 minutes, followed by 10 minutes at 72°C.
Howard's Protocol and Primers for 16s Pyrotagged PCR:
| Pyrotag PCR Master Mix | ||
| Reagent | Amount μl | |
| Water | 39.25 | |
| 10x Buffer | 5 | |
| dNTPs (10mM) | 1 | |
| F primer (10 μM) | 0.5 | |
| R primer (10 μM) | 0.5 | |
| BSA (10mg/mL) | 1.5 | |
| Taq (5u/UL | 0.25 | |
| DNA | 2 |
| Cycling Conditions | |||
| Temp. °C | Time (min) | Cycles | |
| 95 | 3:00 | ||
| 95 | :30 | 25 | |
| 55 | :45 | ||
| 72 | 1:30 | ||
| 72 | 10:00 | ||
| 4 | Hold |
Pyrotag Primers: 5' - emulsion PCR primer/ unique tag/ universal primer - 3'
926 forward primers:
926fA1 : gcc tcc ctc gcg cca tca g agc aaa ctY aaa Kga att gac gg
926fA2 : gcc tcc ctc gcg cca tca g atg aaa ctY aaa Kga att gac gg
926fA3 : gcc tcc ctc gcg cca tca g ctc aaa ctY aaa Kga att gac gg
926fA4 : gcc tcc ctc gcg cca tca g cag aaa ctY aaa Kga att gac gg
926fA5 : gcc tcc ctc gcg cca tca g tac aaa ctY aaa Kga att gac gg
926fA6 : gcc tcc ctc gcg cca tca g tcg aaa ctY aaa Kga att gac gg
926fA7 : gcc tcc ctc gcg cca tca g act aaa ctY aaa Kga att gac gg
926fA8 : gcc tcc ctc gcg cca tca g cgt aaa ctY aaa Kga att gac gg
926fA9 : gcc tcc ctc gcg cca tca g acacg aaa ctY aaa Kga att gac gg
926fA10 : gcc tcc ctc gcg cca tca g tgctg aaa ctY aaa Kga att gac gg
926fA11 : gcc tcc ctc gcg cca tca g cagcaaa aaa ctY aaa Kga att gac gg
926fA12 : gcc tcc ctc gcg cca tca g catgaaa aaa ctY aaa Kga att gac gg
1492 reverse primer:
1492rB1 : gcc ttg cca gcc cgc tca g agc tac ggY tac ctt gtt acg act t
Summary of PCR Conditions
| Reagent | Howard Ochman | EX Takara HS | Sigma Taq | |
| Total Volume | 50 μl | 25 μl | 25 μl | |
| MgCl2 conc. | unknown | 2.5 mM | 2 mM | |
| dNTP conc. | each 0.2mM | each 0.2mM | each 0.2 mM | |
| F primer conc. | 0.1 μM | 0.2 μM | 0.2 μM | |
| R primer conc. | 0.1 μM | 0.2 μM | 0.2 μM | |
| BSA conc. | 0.3mg/ml | 0 | 0 | |
| Taq conc. | 1.25U/reaction | 1.25U/reaction | 1.25U/reaction | |
| Template conc. | 2 μl/50 μl | 1 μl/25 μl | 1 μl/25 μl |
Troubleshooting