PCR Practice:

Create forward and reverse primers that amplify the majority ofthe sequence below. Draw circles around the section of the sequenceyour primers are taken from. Highlight the region of the sequencethat your PCR will amplify. Indicate the 3’ and 5’ ends on yourprimers.

>gi|297828897|ref|XM_002882285.1| Arabidopsis lyrata subsp.lyrata glyceraldehyde-3-phosphate dehydrogenase C subunit (GAPC),mRNA

CTCCACGTTCTTCTCTCTTTTAAATAGACCCTTCACGGACCCTTCTCACTCACCTATCTCACTGTTAAAT

ATCTCTCTGTGAATCTCATCTTCAACCTCTCTTACACACTCGCGTTTTCGATTCAAACAATGGCTGACAA

GAAGATTAAGATCGGAATCAACGGATTCGGAAGAATCGGTCGTTTGGTTGCTAGAGTTGTTCTTCAGAGG

GACGATGTTGAGCTCGTTGCTGTTAACGACCCCTTCATCACCACTGAGTACATGACCTACATGTTCAAGT

ATGACAGTGTTCACGGTCAATGGAAACACAATGAACTCAAGATCAAGGATGAGAAGACCCTTCTCTTCGG

TGAGAAGCCAGTCACTGTTTTCGGCATCAGGAACCCTGAGGATATCCCATGGGCCGAGGCTGGAGCTGAC

TACGTTGTTGAGTCTACCGGTGTCTTCACTGACAAGGACAAGGCTGCTGCTCACTTGAAGGGTGGGGCCA

AGAAGGTTGTCATCTCTGCCCCCAGCAAAGACGCACCCATGTTTGTTGTTGGTGTCAACGAGCACGAATA

CAAGTCCGACCTTGACATTGTCTCCAACGCTAGCTGCACCACTAACTGCCTTGCTCCCCTTGCCAAGGTT

ATCAACGACAGGTTTGGAATTGTTGAAGGTCTTATGACAACAGTCCACTCTATCACTGCTACTCAGAAGA

CTGTTGATGGTCCATCAATGAAGGACTGGAGAGGTGGAAGAGCTGCTTCATTCAACATTATTCCCAGCAG

CACTGGAGCTGCCAAGGCTGTCGGAAAGGTGCTTCCAGCCCTTAACGGAAAGTTGACCGGAATGTCTTTC

CGCGTCCCAACCGTTGATGTCTCAGTTGTTGACCTTACTGTCAGGCTCGAGAAAGCTGCTACCTATGATG

AAATCAAAAAGGCTATCAAGGAGGAATCTGAAGGCAAACTTAAGGGAATCCTTGGATACACCGAGGATGA

TGTTGTCTCAACTGACTTCGTTGGTGACAACAGGTCGAGCATTTTTGACGCCAAGGCTGGAATTGCATTG

AGCGACAAGTTTGTGAAATTGGTGTCATGGTACGACAACGAATGGGGTTACAGTTCCCGAGTGGTCGACT

TGATCGTTCACATGTCAAAGGCCTAAGCTAAGAAGCAGATCTCGAACGGCGAGGAGTGGAAAGTCATCTG

TTCATCCTCTTTTATGGTCTGACTTTGTCGTTTTCGAATAAAATTTCTTTGAACTTGGAACCCTTTTTTT

TTTTTTTTGGTTTTCTTAATTCTCATTCATGTGAGTTGATGGGAGTTTGTAGACCGGTGTTTTACTGAAG

CCCTTTCGTTTTTGGCTTTTGATATATTGAGTTACTTATGGTTTTTCATTTTGTTTCACTTCTCTTTTTT

TCTATATACTAAATCAAATCTGAACGAAC

Forward:_____________________________________________

Reverse:_____________________________________________

- Primers must be 18-30 base pairs in length

- Cannot contain intra-complementary sequences

- Must have a G-C clamp of 2-5 base pairs

- Also include GC content of primers and melting temperature

-Include number of nucleotides in primer

- G/C clamp- write out the G/C clamp in this format: 3' - GCC(whatever the G/C clamp is should go in the place of the GCC)

- Circle the template strand in the location the primers aretaken from