PRIMER SELECTION AND DESIGN
PRIMER DESIGN GUIDELINES
1. Primers should be between 20 and 30 nt in length.
2. Avoid complementarity within and between primers.
3. The GC content should be approximately 50%.
4. Avoid mono- or dinucleotide repetition within primers.
5. The primer should end on a G or a C.
6. Primers should end on the second (or fi rst if necessary) position of a codon.
7. The melting temperatures of primer pairs should be within 5°C of one another.
1. Primers should be between 20 and 30 nt in length.
2. Avoid complementarity within and between primers.
3. The GC content should be approximately 50%.
4. Avoid mono- or dinucleotide repetition within primers.
5. The primer should end on a G or a C.
6. Primers should end on the second (or fi rst if necessary) position of a codon.
7. The melting temperatures of primer pairs should be within 5°C of one another.
Brandon-Mong et al. (2015) compared insect DNA barcoding primers and found high success with mlCOIintF and HCO2198. You can also consult the BOLD primer database for potential primers.
PCR REAGENTS AND SET-UP
In Malaysia we often use Econotaq PLUS GREEN mastermix for our PCR premix. Colleagues in Thailand use AccuStart II PCR SuperMix from Quanta BioScience.
a. Add 1ul of gDNA to each tube containing 24ul PCR mastermix.
For our workshops we use primers LCO1490 and HCO2198 and MLepF and LepR.
b. Centrifuge the tube at 1000xg for 20s and place in the PCR machine.
a. Add 1ul of gDNA to each tube containing 24ul PCR mastermix.
For our workshops we use primers LCO1490 and HCO2198 and MLepF and LepR.
b. Centrifuge the tube at 1000xg for 20s and place in the PCR machine.
A typical PCR recipe is:
PCR THERMOCYCLE PROGRAM
COI Fast PCR program:
Initial denaturation at 94C for 1min.
5 cycles of denaturation at 94C for 30s, annealing at 45C for 40s, and extension at 72C for 1min.
35 cycles of denaturation at 94C for 30s, annealing at 51C for 40s, and extension at 72C for 1min.
Final extension at 72C for 10mins.
Initial denaturation at 94C for 1min.
5 cycles of denaturation at 94C for 30s, annealing at 45C for 40s, and extension at 72C for 1min.
35 cycles of denaturation at 94C for 30s, annealing at 51C for 40s, and extension at 72C for 1min.
Final extension at 72C for 10mins.
PCR CHECK / GEL ELECTROPHORESIS
For comprehensive instructions on how to make the gel and run the electrophoresis see section 3.14 in the DNA barcodes for insects book chapter.