Sep 30, 2024
Barcoding PCR for MiSeq sequencing
- 1Institute of Soil Biology and Biogeochemistry, Biology Centre CAS
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: eva.petrova@bc.cas.cz
Protocol Citation: Eva Petrova, Roey Angel 2024. Barcoding PCR for MiSeq sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3jb7l25z/v1
Manuscript citation:
Rudi K, Rud I, Holck A. A novel multiplex quantitative DNA array based PCR (MQDA‐PCR) for quantification of transgenic maize in food and feed. Nucl Acids Res. 2003;31:e62–e62.
http://nar.oxfordjournals.org/content/31/11/e62
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: May 24, 2018
Last Modified: September 30, 2024
Protocol Integer ID: 12420
Abstract
Barcoding PCR for MiSeq sequencing
Barcode head 8mer-GCT ATG CGC GAG CTG C Modified from Rudi et al. (2003)
PCR mixture
PCR mixture
| Reagent | Final. conc. | 1 tube (50μl) | 1 tube(25μl) | 96 tubes (50μl x100) | |
| PCR H2O | 36.15 | 18.075 | 3615 | ||
| 10X DreamTaq Green Buffer | 1x | 5 | 2.5 | 500 | |
| dNTP mixture (2.0 mM each) | 0.2mM | 5 | 2.5 | 500 | |
| MgCl2 (25 mM) | 2mM | 0* | 0 | 0 | |
| BSA (20 μg/μl) | 0.08 μg/μl | 0.2 | 0.1 | 20 | |
| Barcode (50 μM) | 0.4 μM | 0.4 | 0.2 | 0.4 x 100 | |
| Dream Taq green (5U/μl) | 1.25 U/50μl | 0.25 | 0.125 | 25 | |
| Template | 3 | 1.5 | 3 x 100 | ||
| Final volume | 50 | 25 | 5000 |
* Buffer already contains MgCl2 at a final conc. of 2.0 mM.
PCR program
PCR program
1. 94◦C – 4′
2. x 4 – 9 {
a. 52◦C – 30′′
b. 72◦C – 45′′
c. 94◦C – 30′′
}
3. 52◦C – 30′′
4. 72◦C – 10'