1. Sequence Input

2. Primer Length

3. Melting Temperature (Tm)

4. GC Content

5. GC Clamp

6. Primer-Dimer & Secondary Structure Avoidance

7. Amplicon Size

8. Primer Location Preferences

9. Salt & Mg²⁺ Concentration (Advanced Setting)

10. 3’ End Considerations

11. Specificity Check (BLAST Analysis - Post Primer Design)

Depends on your research question. Talk with your supervisor.

What are you trying to amplify (cDNA or gDNA)? What size amplicon do you want/need? Do you have an existing primer you want to use for one end? Do you need to restrict primer locations to certain parts of the DNA/RNA? Are you trying to avoid a consensus region for a gene family?

Honestly, you can keep most of the defaults. Unless it's a complicated question, I normally only adjust the amplicon size parameters.