Trichoptera PCR primer screening (Limnephilidae)

Knowledge graph centered on Trichoptera PCR primer screening (Limnephilidae) with 18 nodes and 44 connections. Top connected: Lepidoptera, Trichoptera, Limnephilus externus, Asynarchus nigriculus, gene coalescence.

Method synopsis

Systematic testing of 107 published PCR primer pairs from multiple gene regions to identify those that successfully amplify target sequences in caddisfly species. Includes PCR amplification, gel verification, and Sanger sequencing of products.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure as described in the canonical source

Steps below were extracted from the paper that introduces this protocol — Identifying PCR primers to facilitate molecular phylogenetics in Caddisflies (Trichoptera) (2015), Zoological Systematics. Implementations in other papers (listed below) may differ.

1. Test PCR primer pairs
   107 primer pairs for 45 nuclear and 3 mitochondrial genes were tested using primers from published sources. PCR reactions were conducted in 25μL total volume containing 1μL template, 1μL Forward primer, 1μL Reverse primer, 9.5μL distilled deionized water, and either 12.5μL NEB 2X Quickload MasterMix or 12.5μL Top-Taq DNA Polymerase Mastermix.
   Quantities: 107 primer pairs tested, 25μL total PCR reaction volume, 1μL template, 1μL each primer, 9.5μL water, 12.5μL mastermix per reactionDuration: Specific cycling conditions varied by primer pairConditions: PCR amplifications performed in BioCycle MyiCycler, C1000, or S1000 Thermal Cyclers
   Equipment: NEB 2X Quickload MasterMix, Top-Taq DNA Polymerase Mastermix, BioCycle MyiCycler, C1000 Thermal Cycler, S1000 Thermal Cycler
2. Verify PCR products and sequence
   PCR product sizes were verified by agarose gel electrophoresis using 1% agarose slab gels in TAE buffer or on a Qiagen QiAxcel capillary electrophoresis instrument. PCR products of correct size were prepared for sequencing in both directions using Sanger sequencing with ABI Big Dye Terminator chemistry.
   Quantities: 1% agarose gels, individual PCR products sequenced in both directionsDuration: Standard gel electrophoresis and sequencing protocolsConditions: Laboratory conditions for gel electrophoresis and sequencing
   Equipment: 1% agarose slab gels, TAE buffer, Qiagen QiAxcel capillary electrophoresis instrument, ABI Big Dye Terminator sequencing chemistry

Typical Equipment

• NEB 2X Quickload MasterMix
• Top-Taq DNA Polymerase Mastermix
• BioCycle MyiCycler
• C1000 Thermal Cycler
• S1000 Thermal Cycler
• 1% agarose slab gels
• TAE buffer
• Qiagen QiAxcel capillary electrophoresis instrument

Output Measurements

• successful primer pairs
• PCR amplification success rates
• DNA sequences

Papers Using This Protocol (1)

Species Studied With This Protocol