In addition to the 2 μl template from the first round PCR, the reaction composition of the second round PCR was 10 μl 10× Titanium Taq PCR Buffer, 8 μl dNTPs (2.5 mM each), 1 μl Titanium Taq, 5 μl FwdGEX Primer (10 μM), 5 μl RevGEX Primer (10 μM) adjusted to a total of 100 μl with PCR-grade water.
The amplification mix comprised 19 µl of mix and 1 µl of PCR product template from the first amplification round.
Secondary PCR was performed on purified template from the first RT-PCR product, using the same protocol.
In the second reaction, the same master mix was used but with different primer concentration (10 μM of each of the species-specific primers in separate tubes), 2 μL DNA template from the first reaction in a 25 μL reaction volume.
Using the same primer sets as for 15 of the loci in the genomic CAPS analyses, RT-PCR was carried out using 50 ng of template from the first-strand cDNA in a 25 μl volume with 20 ng template, 5× buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.1 mM each primer, and 0.5 unit Taq polymerase (Promega).
Alternatively, download a template from a third-party source.
You can either download a template from a third-party site or use Excel to connect with Microsoft Office Online.
High quality polyA+ RNA was isolated from total RNA as template for first- and second-strand synthesis.
Thus, when users would like to replicate existing environments, they need to describe templates from the first.
Second PCR was done in a 10-μl reaction with 1 μl reaction mix, 0.2 μM primer, and 1 μl PCR product from first PCR as template.
