The mammalian barcoding kit provides DNA-based identification of mammals using materials from individual organisms (feces, tissue, etc.). The kit contains universal mammalian primers, all components of the barcoding PCR master mix (syl1005) and materials for Sanger DNA sequencing (external service). The PCR reactions will be performed on a qPCR machine with an intercalating 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 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.
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 Mammalian barcoding kit 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 (2x)
- Pfu DNA polymerase
- PCR grade water
- Universal mammalian primer mix with an intercalating dye
- Sequencing primer (5µM)
The kit contains materials for 100 reactions.
Equipment and additional kits required
- qPCR machine
- -20 °Freezer
- Fecal sampling set (SYL006)
- DNA isolation kit (SYL002)
- (Prepaid) sequencing barcode labels (for example from Eurofins)
- Sequencing analysis software (e.g., Mega 11)