I am working on a ddRAD-seq experiment and looking for a detailed protocol, particularly focusing on restriction enzyme selection, library preparation, and adapter selection. I would appreciate if anyone could share an optimized protocol or insights on choosing the best restriction enzymes for different genomes. Additionally, any recommendations on adapter design and ligation efficiency would be helpful.
Has anyone encountered challenges in enzyme compatibility or library preparation steps? Any troubleshooting tips would also be valuable.
Vikas Manikrao Shukre
Extract DNA – High-quality (~1–5 µg). Double Digest – Use compatible REs. Adapter Ligation – Add unique barcoded adapters. Size Selection – 200–500 bp (gel or bead-based). PCR Amplification – Enrich target fragments. Library QC & Sequencing – Check size, quantify, and pool for Illumina sequencing.
Restriction Enzyme Selection for ddRAD-seq
- Choose enzyme pairs that provide even genome coverage and suitable fragment sizes (200–500 bp).
- Common pairs:EcoRI + MspI (GAATTC + CCGG) – General use PstI + MspI (CTGCAG + CCGG) – Good for high-repeat genomes SphI + MluCI (GCATGC + ACGCGT) – Alternative for varied GC content
Library Preparation Steps
0 votes
0 thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Zoology
- Ornithology
More Gemini Gajera's questions See All
Similar questions and discussions