To genotype the identified fixed SNP from the genotype–phenotype association analysis (see results) across specimens used in RAD-Seq analysis, genomic DNA was extracted from 83 individuals (42 wildtype and 41 yellow mutants) using thoracic tissue and standard protocols of a Qiagen DNeasy Kit. Primers were designed to amplify the candidate SNP-harboring region (SNP position 3,802,598 on NC_015770.1 identified from RAD-Seq and WGS analysis; Bter_CM1177_3802598_L 5′-CCTCTTTGTCCTTCGCTTGC-3′, Bter_CM1177_3802598_R 5′-CCAGCAAGATTCGCGAAATAGT-3′) and were amplified through PCR (94 °C 2 min, 35 cycles of [94 °C 30 s, 51 °C 30 s, 72 °C 90 s], 72 °C 10 min; 15 µl reaction with 0.3 µl 10 uM primers, 5.9 µl water, 1 µl DNA, and 7.5 µl HotStart Taq Mastermix (NEB)), purified with ExoSap-IT or a Qiagen MinElute PCR Purification Kit, and Sanger sequenced at the Pennsylvania State University Genomics Core Facility (University Park, Pennsylvania, USA). Manual inspection, trimming, and alignment of Sanger sequence data (chromatograms) was conducted in Geneious v. 8.1.9 and the previously identified SNP was manually called.
Hotstart taq mastermix
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
HotStart Taq Mastermix is a ready-to-use solution containing Taq DNA Polymerase, dNTPs, and reaction buffer. The polymerase is inactive at lower temperatures, preventing nonspecific amplification during setup.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using hotstart taq mastermix
1
Genotyping Fixed SNP via Sanger Sequencing
2
Molecular Identification of Anopheles Mosquitoes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Individual mosquitoes were identified to species level according to Santolamazza et al [53] .
PCR reactions contained 2μL of 10μM forward primer (5'-TCGCCTTAGACCTTGCGTTA-3'), 2 μL of 10μM reverse primer (5'-CGCTTCAAGAATTCGAGATAC-3'), 1μL extracted DNA and 10μL HotStart Taq Master Mix (New England Biolabs, UK), for a final reaction volume of 20μL. Prepared reactions were run on a BioRad T100™ thermal cycler with the following conditions: 10 minutes denaturation time at 94°C, followed by 35 amplification cycles of 94°C for 30 seconds, 54°C for 30 seconds and 72°C for 60 seconds, followed by a final extension at 72°C for 10 minutes. PCR products were visualised on 2% E-gel agarose gels in an Invitrogen E-gel iBase Real-Time Transilluminator. A Quick-Load ® 100bp DNA ladder (New England Biolabs, UK) was used to determine band size. Amplified PCR products of 479 bp or 249 bp were indicative of An. coluzzii or An. gambiae s.s., respectively.
As the dominant species, only An. coluzzii individuals of the same age and resistance phenotype were selected and pooled for 16S rRNA sequencing.
PCR reactions contained 2μL of 10μM forward primer (5'-TCGCCTTAGACCTTGCGTTA-3'), 2 μL of 10μM reverse primer (5'-CGCTTCAAGAATTCGAGATAC-3'), 1μL extracted DNA and 10μL HotStart Taq Master Mix (New England Biolabs, UK), for a final reaction volume of 20μL. Prepared reactions were run on a BioRad T100™ thermal cycler with the following conditions: 10 minutes denaturation time at 94°C, followed by 35 amplification cycles of 94°C for 30 seconds, 54°C for 30 seconds and 72°C for 60 seconds, followed by a final extension at 72°C for 10 minutes. PCR products were visualised on 2% E-gel agarose gels in an Invitrogen E-gel iBase Real-Time Transilluminator. A Quick-Load ® 100bp DNA ladder (New England Biolabs, UK) was used to determine band size. Amplified PCR products of 479 bp or 249 bp were indicative of An. coluzzii or An. gambiae s.s., respectively.
As the dominant species, only An. coluzzii individuals of the same age and resistance phenotype were selected and pooled for 16S rRNA sequencing.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!