The DNA (meta)barcoding master mix for environmental DNA metabarcoding and DNA-based identification of individuals. The PCR reactions will be performed on a qPCR machine with a dye with similar to SYBR Green™, followed by a dissociation curve assay for quality control. Alternatively, a regular PCR machine can be used followed by gel electrophoresis as quality control. The DNA barcoding master mix offers excellent performance for sensitive, robust and accurate DNA amplification with a low error rate. The barcode PCR mix is highly resistant to inhibitory factors such as humic acids present in environmental samples. The barcoding master mix is based on a Pfu DNA polymerase from Pyrococcus furiosus with a modified environmental PCR mix containing a dye similar to SYBR Green™
Pfu DNA polymerase has superior thermostability and proofreading properties compared to Taq DNA polymerase. Pfu DNA polymerase possesses 3′ to 5′ exonuclease proofreading activity by removing nucleotides that are mis incorporated. Pfu DNA polymerase results in an error rate of only 1 in 1.3 million base pairs, making it perfect for studies based on (meta) barcoding.
The DNA (meta)barcoding master mix has:
- High resistance to inhibiting factors. Environmental DNA (eDNA) extracts contain often multiple PCR inhibiting factors. Normal PCR master mixes are sensitive to these substances.
- Highest possible sensitivity (1 DNA copy per reaction). Environmental (water) samples may contain very low amounts of target DNA.
- Low Error Rate: The difference between species can be as less as only one base pair. PCR errors can make the difference between one species and the other and should therefore be avoided.
- High amplicon concentration. PCR products used for (next generation) sequencing should have a high amplicon concentration.
- Barcoding PCR mix with a dye similar to SYBR Green™ (2x)
- Pfu DNA polymerase
- PCR grade water
The kit contains materials for 200 reactions.
Equipment and additional kits required
- qPCR machine
- -20 °Freezer
- Environmental DNA sampling kit (SYL009) or Fecal sampling set (SYL006)
- DNA isolation kit (SYL002)