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PCR primer pairs and sequencing primer for each SNP are reported in Table 1.
The expected distribution of amplified fragment sizes, including the primer, for each variable region is shown in Figure 1.
Therefore, contigs were extended using GenomeWalker (Clontech, Mountain View, CA) ligation mediated PCR using a single gene specific primer for each contig end.
For screening for loss of selectable marker a different 3' primer for each transgene was used in combination with the 5' genotyping primer.
The ASPE amplifications were multi-plexed using 500 nmols of primer for each of the 11 loci, 10× ASPE buffer, 0.075 µL of Tsp DNA polymerase, 0.5 µL dNTP 20× mix, 0.125 µL of 400 uM biotin-dCTP, and pooled PCR products as templates.
F: forward primer, R: reverse primer for each respective gene.
One PCR primer for each locus was Cy5-labelled.
The reverse primer for each PCR fragment was biotinylated.
The forward primer for each pair was labeled with fluorochromes (HEX and TET).
The sequences of a suitable primer for each marker gene are given in Table I.
Only one primer for each sequence was used, and 40 thermal cycles were carried out.
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