Help for bisulphite primer design

[jyuems]

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