UCSC In-Silico PCR
  
>chr12:6329057-6329783 727bp TAGACAGTGAGTTTCTCCCGC GCATAGCGTCCCTCATCCTC
TAGACAGTGAGTTTCTCCCGCggctggagacgaggaggctgggggagggc
cgggggagcgcgggaggcgctcccagaggggaccacgagaggcggagggc
gcgggatgcggggcggggcctggggttgccgcccgaggctcaccggcccg
cgtccccgcagctgatgaccccgcgacgctgtacgccgtggtggagaacg
tgcccccgttgcgctggaaggaattcgtgcggcgcctagggctgagcgac
cacgagatcgatcggctggagctgcagaacgggcgctgcctgcgcgaggc
gcaatacagcatgctggcgacctggaggcggcgcacgccgcggcgcgagg
ccacgctggagctgctgggacgcgtgctccgcgacatggacctgctgggc
tgcctggaggacatcgaggaggcgctttgcggccccgccgccctcccgcc
cgcgcccagtcttctcagatgaggctgcgcccctgcgggcagctctaagg
accgtcctgcgagatcgccttccaaccccacttttttctggaaaggaggg
gtcctgcaggggcaagcaggagctagcagccgcctacttggtgctaaccc
ctcgatgtacatagcttttctcagctgcctgcgcgccgccgacagtcagc
gctgtgcgcgcggagagaggtgcgccgtgggctcaagagcctgagtgggt
ggtttgcGAGGATGAGGGACGCTATGC

Primer Melting Temperatures
  Forward: 59.5 C tagacagtgagtttctcccgc
Reverse: 62.1 C gcatagcgtccctcatcctc
The temperature calculations are done assuming 50 mM salt and 50 nM annealing oligo concentration. The code to calculate the melting temp comes from Primer3, the formula by Rychlik W, Spencer WJ and Rhoads RE NAR 1990, which can be activated in Primer3 with PRIMER_TM_FORMULA=0.

Help
  What is chr_alt & chr_fix?
Replicating in-Silico PCR results on local machine