preselect

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In the second round of PCR, selective amplification was achieved using the MseI+CAG and EcoRI+AAC (fluorophore NED [yellow] primers); each selective primer has an identical sequence to its corresponding preselective primer but with an additional two bases.
For the selective amplification, the reactions were set up as follows: 2[micro]l of 5-fold diluted preselective amplification reaction product, 1.
Primer and adaptor oligonucleotide sequences used in the study Primer or adaptor oligonucelotide Sequence (5[right arrow]3') rDNA primers SR6R AAGTARAAGTCGTAACAAGG LR1 GGTTGGTTTCTTTTCCT M13 fingerprinting primer Phage M 13 GAGGGTGGCGGTTCT EcoRI adapters EA1 CTCGTAGACTGCGTACC EA2 CATCTGACGCATGGTTAA MseI adapters MA1 GACGATGAGTCCTGAG MA2 TACTCAGGACTCAT Preselective primers EcoRI-T GACTGCGTACCAATTCT EcoRI-G GACTGCGTACCAATTCG MseI-C GATGAGTCCTGAGTAAC MseI-G GATGAGTCCTGAGTAAG Selective primers EcoRI-TG 6 FAM-GACTGCGTACCAATTCTG EcoRI-GT 6 FAM-GACTGCGTACCAATTCGT MseI-CA GATGAGTCCTGAGTAACA MseI-GT GATGAGTCCTGAGTAAGT Table 2.
AFLP adapters and preselective and selective primers were purchased from Integrated DNA Technologies (Coralville, IA).
Ligation product was diluted 20-fold and used as a template for preselective amplification.
Preselective amplification was performed using specific primers complementary to each adaptor sequence.