Old yellow enzyme was first isolated from brewer\'s bottom yeast (Saccharomyces

Question

Old yellow enzyme was first isolated from brewer's bottom yeast (Saccharomyces calsbergensis) in 1931. It was the first protein discovered to have a non-covalently bound cofactor (FMN) and the protein has been studied extensively as a model flavoprotein for many years. The gene encoding old yellow enzyme was cloned and sequenced by Saito et al. Give the sequences of the primers (forward and reverse) that would amplify exactly the coding region of the gene. Note that the coding region begins at nucleotide 349 and ends at nucleotide 1551. Your primers should also place an NdeI site that overlaps with the start codon of the gene and an EcoRI restriction enzyme site immediately following the stop codon. Hint: PCR primers should have a melting temperature (Tm) value of 72 degrees C. For short DNA duplexes, the following formula is accurate: Tm = (number of G-C base pairs) X 4 degrees C + (number of A-T base pairs) X 2 degrees C. (5 points for each primer).

http://www.ncbi.nlm.nih.gov/nuccore/X53597.1?ordinalpos=2&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum

Explanation / Answer

1141 CAATATGCTT ACGTTGCTGG TGAATTAGAA AAGAGAGCTA AAGCCGGAAA ACGTTTAGCT

1201 TTTGTTCATT TGGTTGAACC TCGTGTAACT AACCCATTCT TGACTGAAGG GGAGGGTGAA

1261 TACGAAGGAG GTAGCAACGA TTTTGTTTAC TCCATCTGGA AGGGCCCAGT CATTAGAGCT

1321 GGTAATTTTG CTCTCCACCC AGAAGTCGTT AGAGAAGAAG

          UAG

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