Primer Generation

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Primer General Requirements:

Sanger Sequencing Primer:

Example of good primer: GCATACTATCATTCGGGG

• [x] 18-24 nucleotides
• [x] For longer nucleotide primers: start with C or G
• [ ] No hairpin – no complementary nucleotide
• [ ] Avoid secondary structure (ex. Hairpin) – no complementary nucleotides within the sequences
• [x] Melting temperature (Tm): try to have lower than 46C
  • Lower the melting temperature the better? Will need to test this out
  • Preferably under the melting temp of thermostable TdT, which is 46C
• [x] 40-60% GC content
• [x] Ending in = GGGG

TdT Nucleotide Extension Primer:

Example of good primer: ACGGG

• [ ] 5 nucleotides
• [ ] Ending in GGG

Dry Lab: Please Generate the following

• [x] Two 5nt primer
• [x] One 15nt primer
  • Please have this primer melting temperature below 46C
• [x] Two 18nt primer
  • 1 below 46C (if possible), 1 above 46C
• [x] One 20nt primer
• [x] One 24nt primer

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15 nt primer, below 46C

GGATTTACTTTGGGG

GAAGTAAAATGGGGG

CAATAGCATGAGGGG

GAATGTCATAAGGGG

CTAAGACATGTGGGG

GAAACAAAGGTGGGG

GCAGCAAAATAGGGG

GAAATGGGAAAGGGG

GATTATGCATGGGGG

CGCAATCATAAGGGG

CATACATATTAGGGG

CAGACGAATTTGGGG

CTATAAAGTCCGGGG

CGTTGTTCATTGGGG

CCAATTAACGAGGGG

CAATCGTATGTGGGG

CAATTGCAATCGGGG

CAGCTATCTTTGGGG

CTTATAGTCACGGGG

GTCGAAGATTAGGGG

GTCAATGTTTGGGGG

CCTTATTGAATGGGG

CTAGTTTCAGTGGGG

CGATAGTTAACGGGG

CATAACTGTTGGGGG

GGAAAATATCAGGGG

GTCTTTAACTCGGGG

CATTTGTAGTGGGGG

GTAGTTTTATTGGGG

GGAATTCCAATGGGG

GCTTATGAACTGGGG

CTTGATTTAGCGGGG

GATTATGCGAAGGGG

GTTACTCATGTGGGG

CGAAAATAGACGGGG

GGAACGATATTGGGG

CTATTTGCAGAGGGG

GCTTTATTATGGGGG

CACACAAACTTGGGG

GTTTACTTATTGGGG

GTCTGTATATCGGGG

CTTTTTCTCCTGGGG

GTAATATCGCTGGGG

CCTAATCTTATGGGG

CTCAATAAACTGGGG

CAATAAAAAGCGGGG

GATATGGATACGGGG

CATAATAAGCGGGGG

GTTCATTTATAGGGG

CAAACTTAGTGGGGG

GTTGCTATTAAGGGG

GAAAATTGCACGGGG

CCTACTTTAGTGGGG

GACTTATAGAGGGGG

CAACATATTCGGGGG

GTAAGTTTGTTGGGG

CTCCATATATCGGGG

GAAAGGCAATAGGGG

CTATGACTATGGGGG

CATGTTGTATCGGGG

GGTTAAACAATGGGG

CGTCAAAGTTTGGGG

CTTTCACTACAGGGG

GTTTACATTTGGGGG

GTTACGATATAGGGG

CGTTATTTCTGGGGG

CGTTTACACAAGGGG

GATGTGTATAGGGGG

GGTTAATCATCGGGG

GCTTACCTTTTGGGG

GTCTCACAATAGGGG

CAGATATTACAGGGG

CCGTCTATATTGGGG

GGACTACTAAAGGGG

CGATAGTTTACGGGG

CTTCTACTCTTGGGG

CATGTGATACTGGGG

CGTCTTTACAAGGGG

GTCTTTAACAGGGGG

GTAATAGCGATGGGG

GCATGTTAAACGGGG

CTAATTCACAAGGGG

CACACGAAAAAGGGG

GTAACAAGGTTGGGG

CATGAGAATTCGGGG

CTTTCCTATGTGGGG

GCTTCCATTTAGGGG

GTTCTAACATGGGGG

CAATAAATCCGGGGG

GAATTTCTGTGGGGG

CTATCTTTCTTGGGG

GTTAAGGGTTTGGGG

GATAAAACTAGGGGG

GTTGTGTAAAGGGGG

CAATGTGTTATGGGG

[lhao03]

18 nt, below 46

CTGACTTATCCAATGGGG

GCTTTAGTCTACTGGGGG

GATAGAGAACTTAAGGGG

CGTTGTAATGACTCGGGG

GATGGTACCTCTATGGGG

CACTTGTTGTTGTTGGGG

CTACTGATCTGTCTGGGG

CTTGCTTAGTAGATGGGG

CAAACTAACATAGCGGGG

CGAGTTATTTCCTTGGGG

GGATTGGATGAAGAGGGG

GCCTAAAGTCAGTTGGGG

CAATAAGACTGAGAGGGG

CTAATGTACGACACGGGG

GTCCTTATAGCGATGGGG

CAGTATAGGTGACAGGGG

CGTAAAAGACATAGGGGG

GTCTATTCTAGTCAGGGG

GAAAGTTTACCGCAGGGG

CTTTTGAATCTAGGGGGG

GAGTACTCACGATAGGGG

CAATAGTGAGTGCAGGGG

CATAAGCGCCAATTGGGG

CTGAAACGACTTGTGGGG

CCAAACGAAACTACGGGG

CGGAAAAACACTAAGGGG

CACAATTAACAGCCGGGG

GCGACAATTTCAAGGGGG

GCCAATGATTTTCGGGGG

GAGGGTAAACAAAGGGGG

CTTTCGTCACTTGTGGGG

GTGAGGATAAATACGGGG

GGTCTAGAATTCCTGGGG

CACCTGGTATTTTTGGGG

GTTCTGTATAATTGGGGG

GATTATTCCAACCGGGGG

GAAAATATCAGCCTGGGG

CATTATGCAACATCGGGG

GTGAAGTTCTTTTTGGGG

GAGCAATTTTTACTGGGG

CTCGACATAAGCTAGGGG

GTGCTTAAAGCTTGGGGG

GCTCATTATTCTAAGGGG

CCGATACTAAGTCTGGGG

GGACTCTGAATGAAGGGG

GATAGTTAGGGTCAGGGG

GAAGAAGGTTTTAAGGGG

GCTGTATTTGTCTAGGGG

CTAGATTCAAACTGGGGG

GTGAGTGATGTCTAGGGG

CAGATTTAACCTTGGGGG

GGAAATTCACAGGTGGGG

CAATCTAATAAACCGGGG

GCAAAAATATCAGCGGGG

CATGGATTATAGTCGGGG

CAAAACAGTCTCAGGGGG

CAATGTGATATAGCGGGG

CTTTTGACCTTTCGGGGG

CATATAGCAAGTTCGGGG

CTGAGGAATGATCTGGGG

GCTTAAACTCTATAGGGG

GGAATTGATGACCTGGGG

GTTCAGTTTTGGGTGGGG

GATTGGCTATTAAAGGGG

GTTGCACGTTAGAAGGGG

GGTGGTTGTTGATAGGGG

GGAGCTAACAAAGTGGGG

GATCTATCAGCTTCGGGG

CTAAGGCGAAAATCGGGG

GAACACTCGGATTAGGGG

CCGACAACTTCAAAGGGG

CTAGCACTAAAGCAGGGG

CGTTAGAGTATTGAGGGG

CAGTTGACACGTAAGGGG

GAGTGCTATGAGATGGGG

CTGTATGAAATGATGGGG

CTAACAGCATACATGGGG

GTGTGTTTAATTCGGGGG

CCGAATTATACTGGGGGG

CGGAATTCCTTTGAGGGG

GCTTATGATTGTTCGGGG

CAATTGACTGTCGTGGGG

GTGATGAATTTGCCGGGG

GATGAAAAATGTCCGGGG

GGCGTTTATACAGAGGGG

GTACTAGCAGAAATGGGG

GACACTATTTAGAAGGGG

GCATGCATTGATAAGGGG

GGAGCAGTAAAAGAGGGG

CTCAAACAATTCTCGGGG

CGGCTGCATAATTTGGGG

CTATCCATTGAACCGGGG

GCTGATTCATATTGGGGG

CAAGTCCATAAACGGGGG

GCGTGAATAAGATAGGGG

[lhao03]

18 nt, above 46C

GGATCCTTTAATTTGGGG

CGAGTTACTCTAAGGGGG

GATACTTAAGTCTCGGGG

GACTGAGTTACTCTGGGG

GGTGTTATGAGCTTGGGG

GCAATTCATACGTGGGGG

GTAGAACAAGAACAGGGG

GTTAGCACTTTACAGGGG

CCTACTACTTATGCGGGG

GTGTAAGTTTCAACGGGG

GTGATGTCAAATGGGGGG

CATAGGCTTCATCTGGGG

GCTTACTTATGCCAGGGG

CTTTTAGGGATTGAGGGG

CTTTAAATAGCCATGGGG

CGCAATGCTATAGAGGGG

GTAACTAAGCTCACGGGG

GGATTATTGACCACGGGG

GAAGAGCCTCAATTGGGG

GGTGAGCAAGATTAGGGG

CTCATTTAAACTCGGGGG

GGTTACTGTTTTGCGGGG

CTATAACGCATCCTGGGG

CACAATGACGCTATGGGG

GTTTATGGTTTGGGGGGG

GCGATGAATTCACAGGGG

GTTTTTGCTTCGTAGGGG

GAAGCTAGAAGTAAGGGG

GTCGACATTTTTAGGGGG

GAATAAAGAGCTGTGGGG

GTCAGGATATCGTAGGGG

GAGTTAAAATATGCGGGG

GACTGCATACAATGGGGG

GACATGACGGAAAAGGGG

CCTTTACAACTCGTGGGG

GGATGACGAATGAAGGGG

GAGCGTTGCATTATGGGG

CCAAAGCATAAACCGGGG

GTCCATAGATACTAGGGG

GTGTCGAGATAATGGGGG

GAAGTAACATTGAGGGGG

GGAAGTCAGAAATTGGGG

CGAGTATCATAAGGGGGG

CTGTTCTGGTGATTGGGG

GCAGGGAATGATAAGGGG

GTTACAAATGTCTAGGGG

CGACAAAAAGTTTAGGGG

GTTTATCTGAGGAGGGGG

CTCTATCTTGATCAGGGG

GGGGCATTATTTAAGGGG

GCGGATCTTTCATAGGGG

GCTAAATCTCTTGAGGGG

CAAACTGAAAACATGGGG

CTCGGTTACCAAATGGGG

CAACGTGATACAATGGGG

GTTTCCGTTTAGATGGGG

CAATCATTGGAGAAGGGG

GCTGTCTAAATACGGGGG

CTTGACTAGAGGTTGGGG

GATTGTTAAACCCAGGGG

GGGTCATTTTATTAGGGG

GTGCATATATACGGGGGG

GTTTGGACTATGCAGGGG

GGGGTTTTAATTCTGGGG

CGTAGTACTTAACAGGGG

CTTTTAGTGGTAGGGGGG

CAATCTGAACTTCTGGGG

GAGCAAAATAGACGGGGG

CGAATATCAAGACGGGGG

GGAAAAAAATCGCCGGGG

GCAGTCGATTGAATGGGG

CATGAGACTGATTGGGGG

GTTGATATCTACGCGGGG

CATACAACACGAACGGGG

GACATGAACATCAAGGGG

CGACAGTAATGGATGGGG

GACGTTAAAACATGGGGG

GTGTGTCGTTTATTGGGG

GTTCGTTAGACGTTGGGG

CAATACACTCGTCTGGGG

GTATCGACTTATTTGGGG

GTCTTGTTCTGTTTGGGG

GAGTTCACTTCAATGGGG

CATATCGCAAAAACGGGG

CATAATATGAGCGTGGGG

CATGCATTTTATTGGGGG

GCTTTAATAACCAGGGGG

CATGCTACTTTTGTGGGG

CGTATACTATGAGCGGGG

CTTAAATGCCATTGGGGG

CGCGTAAAACTAGAGGGG

CTGAAAGCCTCTTAGGGG

CCGGTGAATAGAAAGGGG

CATGATAGACAGGTGGGG

CATGAGGATAAGCAGGGG

GTTAGCATCAGTTTGGGG

CTAATTTAAGTGGAGGGG

GCTCGATTATCAAAGGGG

GTCAAAGTGTAAAGGGGG

CTTGGTTTATGGTTGGGG

CGAAGTGAGAAACAGGGG

CAGCTTAAGATAGGGGGG

GTTCAATGTTATCGGGGG

CACTAACCGAGAAAGGGG

GTGGCTTAAATACGGGGG

CTTTTTCGCCTTCTGGGG

GAGTGAATGATACGGGGG

CGAACAGGGATATTGGGG

CTCATTGCATAGTGGGGG

CATATCCTACTACCGGGG

[lhao03]

5 nt

GGGGG

CGGGG

TGGGG

TGGGG

TGGGG

TGGGG

AGGGG

GGGGG

CGGGG

TGGGG

AGGGG

CGGGG

GGGGG

AGGGG

TGGGG

CGGGG

TGGGG

GGGGG

TGGGG

TGGGG

CGGGG

TGGGG

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