We also amplified the same gene sequence using original primers G1-C12.5.1 and G1-C12.3.2 from A. jeffersonianum, A. texanum, and A. tigrinum, which served as the outgroups for phylogenetic construction.
DNA quantitation followed by PCR, using the original primers and conditions (as above) with the addition of 0.1 mM of each deoxynucleoside triphosphate per reaction, was undertaken using the plasmid plus insert as template.
Using the pre-amplified PCR products as new DNA templates, exons 9 and 20 were separately amplified for sequencing, amplifying exon 9 using the original primers and exon 20 with internal primers.
Following the PCR amplification using the original primers and PCR conditions, the products were ligated into pGEMT-Easy vector (Promega; purchased from VWR Canlab, Mississauga, ON, Canada) and cloned using Top 10 Competent cells (Amersham Biosciences).
Both DNA strands were sequenced using the original primers and a 10 μl reaction mix containing 2 μl of BigDye Terminator Mix (BDTM) version 3.1 (Applied Biosystems, USA).
Using the ABI 7700 Prism Sequence Detector (PE Applied Biosystems) and our original primers and probes, we could quantify the target gene, and minimise amplifying inappropriate genes.
Gaps in the assembly were filled by primer-walking using original PCR amplifications as templates.
We used the original primers G1-C12.5.1 and G1-C12.3.2 [ 43] to amplify this sequence in all five sexual species (LL, JJ, TiTi, TT, and BB) that are involved in unisexual Ambystoma reproduction and got amplifications in all but A. barbouri (BB).
Both strands were screened using the original primers.
The extracted gel mix was heated at 90°C for 10 min, and 4 μl of the solution was taken as the DNA template for re-amplifying by PCR using the original primers without a GC clamp.
