STEP 3: INITIAL PRIMER TESTING

Primers (from Step 1) predicted to amplify SSRs are initially tested on agarose gels to confirm their amplification of a single fragment under standard conditions (58⁰C, 1.5 mM Mg⁺², and 30 cycles).

Approximately 80% of the primer sets amplify unique bands under standard conditions.

Sample Ethidium bromide-stained agarose gel containing 26 primer amplifications.
Outside lanes are size standards. On this gel, 6 of 26 primer pairs did not
amplify any DNA fragments and 4 amplified multiple bands.