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Old 08-01-2014, 05:24 AM   #1
jyuems
Junior Member
 
Location: Finland

Join Date: May 2014
Posts: 6
Default Help for bisulphite primer design

Hi!

I'm studying the methylation state of two genes and could you give me any advice why I can't find proper bisulphite primers for two upstream sequences of genes, regions contain CpG sites and a CpG island. The product should be around 70-80 bp and 2 degree Tm difference, I would also need to have multiple primers that cover most of the CpG site areas. I'm a beginner with this and I can't understand why this is so difficult, the primers I get have a lot of self-dimers (under -4), repeats and runs. Thank you for any help!

Oxtr:

ATGCTGGGGCTGATCCTGTCCCAAATACAATCTCAAAGCAAGAAGAGAACAGTCTTTGCCTCCTACCATTAGCTAATGCTCAGGGAATCCCCACGCCACCTTGGCATGCTGTCACGTAGAACCCGGGAGCAGGCCCTGGAAGATTCTGCCCCTAGGGAATCTTGGCACAGTGCTGTAATTGTGCATTCAGGAAACACTGCCCTTGGCTCATTTAATTCTCGCCACATAATTAGAAGAGGGAGAAATGAAATGTCAGGAGGAAATACACATTTAATTCATGTTAAAGATCCCCGTTCATTTTTTAATCCTGGCTCTTTCTGTTCCTGCCTCGGTCCTTTGTCACCGTAAATCAGTTAAAAAATGATTTCAAAATAGAAAAGGACTGGGGAGTGGAGGAGCAGCCCTTCACAGTTCATCTTCCTCTGGCTCCAGCTAGGATTTTAATTTTGAAGCCTGTGAGGGGACAGAGGTAGCCTTTGGCCCACTGAGAAGCAGAATAATCTTAAATGGTGATAGACTTGGAGCGATGGGGTGGGTGTGTAAGATTTCCATCACTAACTACCTAAAAAATGAAGATTTCTCTCAGGGTTGGTGGGGGGTGGTGGTGGAAGAGTAGGCATTTTCAGTTCCCCTTAATGGGAATATGAACAAAGCCATTCCAGAGCAAGGGCTTATACGAGCTCTCCTAAAAGAATGTGAAGAAAAGGGAAGAAATAAATANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGAGAGAGAGAGAGAAAGAGAGAGCGAGAGCGCGAGCGCGAGCTGAAATGTGACTTCATTAAAATTTACTCTGGGGGAAAAAAGTCACTCTACAAGGTTCCTACATCTCTGAGAGCATCAAGAGCTAAAGCTAGAAAGGAATAAGCTAGGTAGGTTATTCGCGAGCCACTATGAAATAAATCTGAGAGACTAACATTGGGTGAGCTGACCTGGCTCTCTGGGATCGCCGCGACCGGCTTGATCTGCCCCCTAGCGGACATATGTGGTGCTGCAGTCCTGGACTGTGGGAAAGCCGGCCACCTTCAGACAGCGCCGCTTGCTGACGCGGTCCGCGCACAGCCTGGCCACCGCTCTCCGCTTGCCTTCGGTCCGCATTCAGCCAACAGCAAAGCCACAGACTAGAGGACTGCAAAGACGCCGGGATCTACTCGTGGGTGGCACGGAGCGTGGCGCCCGCAGCTCTCCCATCCTGTGCGTAGAGCCCGCGCATCTTTGAGGCCATCTGCTGCGGTGCAACTTCCCCGGTGGCAAGGCAACTTTGGGCTAACCTGCAGGCTCTTTCCAGGTGGCTGGGTCCTGCGCACCGCACCTTGGGGACAGGATGGCTAGTACCAGTGCCACCATCAGTTTCTGAGGGAAGGCTCTGGCGCCTCTCGCACTTCTGGTTCCCGAAGGCGCTCAGGGTACTCCAAGGGACTGAAAAGTGACAGTTCGGACGACGACGCAGTAGAGCTTTGAGGTTGCTGGGTTAGGGGTTTCCTGCGAGAGGCGGCTACCCCAGGAGTTTCTCTCATAGCAGCCCTGCGTCCTGGGGTGCTAGGCTCGCAGGCAGGAGTCCCTCCCTCCGGGAGCCGGAGCTAAGTGTCTTGGATGTGAAGAAGGTGAAGAAGGATGTGCTTGAGCTGAGCTTTTGGGGAACCCCAGGAAGATGTACCTGGAGTAAAGCTTACCAAGAAGGGGGCTGTCATGGGTGCCCATTAGGAGGTGGCTGTGTTAGTGTCCTGAGGGGAGTGGTGTAGGTTTGGGGGCTCTCCGGAGGGATTGGGGCTAGGAGAGGGGTCCCTGCTCTCGGGAAAGCGATTAATCGCGAGCCTCCTCTCTCCGCAGGTGGAGCGCCTGACACTTTCCCGAGATCCACGCTCAGGCGCATCAAGAGAGGATCAAAGAAGGTCTGCATCTTGCAAAGACTCAAAGGAGATCCGGCGAACCGGGATTTCTGTGGTGGTGGCTGAGCAGCCCGGTGGCTGGGTTTGTGTCCTAAGACCCAGGTAACTTCAGATTGCCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGAAGGGGCCAGGCACGCCCCACACGCGTCCTAAGAGAAGCACAAAGGTCTTTTCATTGCCCTCTCGGTTTGTTTCAGGGCAGAAAGCGGCAGATCAGTGCCCGGGTgctcCCGCAGACACCTGGACGGTGCATCGGGCCCCGCCCCACGAGCCACGCTTTAAAGAGCTGCAAGGCCGGGCGCTCCCTCGCGGTCGCGGTCACGGTCATGGAGGGCACCCCCGCAGCCAACTGGAGCATCGAGTTGGACCTCG


Avpr1a:

CGTTCTTAAGAAACTTAAATTCCTTTCCTTGTATCTATGAAGTGTGTGTTCCTCTTTAGAAACAGCGCCCCCTGCTGTCCTTGGTGGTGTCCCTTCGCCAGAGTTAAGACAGTCGGTGGAGTCTGTAGGTGTTTCTGTCCCCTTGAAGCTCCACAACTGGACTCAGTTGTCCAGAGCAAACAATCTTTACTATGGGAGTGGTTGGGGTAGCTTTCGACTCTTCCTTCCAAATGCCTGTTTCTCTCTCCCTAGCTCCTAGTTTAAAAGCCCAGTCGGACAGACAGACAGACAGACAAACAGAACCCCCCCCCACGGATAGACAACACCACTGTGAAAAAGATAGATACAGACAGAAACACACTCACTCACTCACACACACACACACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCACACACACACAGATAGACAACACCAACTGTGAGAGAGATAGCAGACAAGACAAACACACACACACACACACACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACAGACAAATCGTGCCGCCCAGTTCCCACCATCCTGCCGCTGTGGGAACCCGGCTTTCTAGAGTAACGCCTGTCATCCTCACTGGTTCCCGCTGGCCGCGTGGAATAACCAAGGTAGATTATACGCGCCCCGCCGGAAGAGGGAAGGGTGCAAGTAGGTGGGTCTTATCTGTTCGCGAGCAAAGGCAGGGTGACGAAGTCACCAGAAAGAGAGATCCATTTAAGTCACGCGGTTTCTACTCCCAGACAACGTGTAGCAGGAAACCAAGCAAGAGCAAGCAGGGAATAAAATCGGCGTGAGCGGTAGCCTAAGCGCGGAGCTGGGGAAACACTCGGTCTGGGGGACCAGGCAGACCCCAAGTTCAGGTGCACTGAGTTCGCAGTCAGCGGCAGTAGACGCAGATCTCTAAGGACACATTTGCACTTGTTGATGTCCTGCGGGGCTTTAAATTGACTTGTAAAAAGGGGGTTGGAGAAAACCCTGAGGAGCCTAGAGAAGTTTGGGACAGAGCGGGAAAGCGCCTGGCTCTTTCAGGAGGCTGGAGAAATTCAGGAGGCTCAGCTCCACCCCTGCAGCAAGAGAAGAGGACAAATCACAAACGACTCGGAAGGGTGGGGGTTAGAGGTGTAGGGGCAGTCTCCAGCCAGTAGGACTTGAGGGATCGCGTCCTGAGACTGCCAAAGAGTGTATCGGGAGCGCGGAGTGTTGGAAGCTCTCGGAACTAGCAGGAGCGGGGCCACGCACACGTCGGACCCCAAACAGCTCGCCCGGCGCTCGTGGGATTGTACAGCCGAGAGTTCAAAAGGAGGAAGGACAGCGCGCGGAGGAGCTTAGGAGAGGGTTTCTTGGAGGCGGGAACGAAGCAGTCTACGCGGGACAGGGCGTCACGGGCGCTCCCCCGACAGCATGAGTTTCCGGAGAGATTCCTATGA
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