How to design VN primer to attach with universal reverse primer
Follow this link will give an idea about your questions
https://www.twistbioscience.com/products/oligopools?tab=oligo-pools&utm_source=google&utm_medium=cpc&utm_campaign=PSR-GLBL-FY21-1785-OLIGO-Twist-Oligos-Product&adgroup=114820678223&utm_term=primer%20synthesis&utm_content=kwd-126519194&creative=707262899505&device=c&matchtype=b&location=9112435&gad_source=1&gclid=CjwKCAjwk8e1BhALEiwAc8MHiDZFh0-Ag2xpASMR85dZjciOLI4i7wIBXpj6Q-k5Ix0QzF5ss7y_7RoCXVoQAvD_BwE
Also read this article is very helpful
Article MicroRNAs Specific Primer Design using miRNA Design Tool
Steps for Designing VN Primers for miRNA:
1-Identify Target miRNA Sequences
2-Select Regions for Primer Binding
3-Design the Forward VN Primer
4-Design the Universal Reverse Primer
5-Check Specificity: Use tools like BLAST to check the specificity of your designed primers against the genomic or transcriptomic
6-Consider Melting Temperature (Tm): ideally within 2-5°C of each other
7-Optimize PCR Conditions: After designing the primers, optimize the PCR conditions (annealing temperature, extension time) for best results.
Tools for Primer Design:
Primer3: A widely used tool for designing primers.
OligoCalc : For calculating Tm and other properties.
UCSC Genome Browser: To check for conservation and specificity.
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