MSP primers not working - (Sep/04/2005 )
I use these primer for MSP, one for methylated and other for unmethylated
When i do PCR, i cann't see any bands.
methylated primer:
forword 5'-AGC GTT GCG GAA GTA CGC GG-3'
reverse 5'-AAA CCC GCC CCG ACG CCG CC-3'
unmethylated primer:
forword 5'-AGT GTT GTG GAA GTA TGT GG-3'
reverse 5'-AAA CCC ACC CCA ACA CCA CC-3'
and my dna sequense
GGGGAGGCGGACGTTGCAGTGAGCCGAGATTGTGCCACTGCACTCCAGCCTGGGTGACAGACCGAGACTC
CGTCTCAAAAAAAAAAAAAAAAAAATATGGCTGGGCGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGG
AAGGATGAGGTGGGAGGACCCCTTGAACCCAGAAGCCCAGCAAAACCTTGTCTTTAAAAAAAAAAAAGAA
CTGTGCACAAAGATTTCAGAGAGTGCTAAAGATTAGCGCATGGATAAGGAAGTTCTGTGAAGAGTTGAAG
TGCTAGGTGAAGAGGTGGCACGGGGGAGGAGGGGGCGGAAGGGGAGAAAGGGTGTCACGCTTCATAACGG
TCTCCAAACCTCTTTGTCCAGGAGGAAATGAAGTCATCTGTCCTCTCAGCAATCAGCATGACAGCCTCCA
GCCAAGTAACCCTGGAGTCATGAGAGCTGCTAGGGGAGCAACATGAATCATGACGCGCCCCCTGGGAATT
TCCTGATTACTAACCTGGGAGTTTCGGGGTAAGTCCTCAGGCTGCAGCATCTCTGTTTATGTTCTGGTCA
CGTTTATTTACAATTAATGGGTTCTCAAATCCAAACAAAACTGACCACAGTCTTCTAGAGGAAGTAGCAA
GGTTGGCTCTGAAGCCTATAGCATCTCTGACTCAGTCTGTCCCCTGGAAGGCTGGCAGCTCAGCAAGCAC
AGAAGTCTCTCCAGAAGACAGTGGGTCACCTGCCTCCCAAAAGCTGAAAGACTAACTTGTAATTTCCCCA
GCAGGCAGCTGGGATCCTGAGCCCTCGGCTGGGGCAGAGCAAAGGAGCCTTCCTCCTTCCTACCTTCCTG
GCACTCTCCCTGCCTTCCTTCTGTCACTCTCAGGTGGACCCAGACCCAAGGTCCAGATTTGCAAGGCAGG
AAAATGCTGCAGGCCTAGGCTGGGAAAGGGCCCAAAGCCGCTAGTGGATTGCTGGGACTCAGCCTCCTCC
TTCCCACTAAGAGAGCGAGTCGTACTGGGTTCAAAATGACCCCANNCCCTGGTTTCTGACACTAGGGGAA
AGAGATGGGCGTGACAGAATCACAGAANCCCTGCTATGTTCGTCCAAGTGTGCCCAGAGATGCGTGTGTG
TGTGTGTGTATACACAAATGTCTGCTTATCCTCAGGCAGGAAGGGTGGATNCAGTCATTTACACATGGTC
TGTTTTTCTGGAGGACAATTTTATTTGATAAACAATTGTTTCTATCTGAATAGAATAAACAAGGCTCTAT
GATGAAGTAAAACACTAAATACACATGCATTAAAAAATGCATAATTATCTTTTTGGAATGGGCTATACAG
AGATGTGCTTTTTAACCTGTTAAGAGTGTAAAAGGACAAACAGTGAAAANTNATNCNTCCTCTTANTTTG
TCCTCCAGTCTCCCANTTCCTCTACTCAGNGGTGAGNGAGNCTTCCACACCCCTCCAGAACCTCCACAGT
TAGAACTGTCTACATGTTTCCCATTGCTTTTACTTTTATTCTTGCCTGCACANATAAATGAATTGCTCCA
TTATGGAAACTTCCCAAAACGCATCCGCGTTAACACTTCAATAGGAAGCACCAACAGTTTATGCCCTAGG
CTTTGTTCCCACAATCCTGTAACATCATATCACGACACCTAACCCAATCCTTATCAAGCCCTGTCAAAAA
CGGACTTTAAACCAAGCTGCAAATTTTCAGTAATCTGGCCTTGCCTTTCCCCCTCTGATAGCACCATCAA
ACAAACCCCCTTACTGCCGAAAGCAATAAGCCCGCCTTTGTTCCATCCACTGGTTGTGTTGGTGATATCT
GGGGACTGCCACTGAACAGACGCACAGAGGGAGCCCCTACAGGCAGGGGTTTTTCTGTCTGTGCTTCTTG
GGAGAGTATGTCTCGTACATTTGTCGCGTGATGAAGACTTCACAGCTCCATCAGCTGCGGGCAAGGGGGT
CTGAGGCAGTCTTAGGCAAGTTGGGGCCCAGCGGGAGAAGTTGCAGAAGAACTGATTAGAGGACCCCAGG
AGGCTTCAGAGCTGGGCGAGGTAGAGAGTCTCCTGTGCGCCTTCTCTCCTCTCTGCAATTCGGGGACTCC
TTGCACTGGGGCAGGCCCCCGGCAGGTGCATGGGAGGAAGCACGGAGAATTTACAAGCCTCTCGATTCCT
CAGTCCAGACGCTGTTGGGTCCCCTCCGCTGGAGATCGCGCTTCCCCCAAATCTTTGTGAGCGTTGCGGA
AGCACGCGGGGTCCGGGTCGCTGAGCGCTGCAAGACAGGGGAGGGAGCCGGGCGGGAGAGGGAGGGGCGG
CGCCGGGGCGGGCCCTGATATAGAGCAGGCGCCGCGGGTCGCAGCACAGTCGGAGACCGCAGCCCGGAGC
CC
Can you suggest me? These primers work or not?
Thanks for you help
Hi,
No bands doesn't necessarily mean the primers are bad, but there are apparent problems with your primers.
First, the sequence you provided has many sequence errors, you should use a complete genomic sequence for primer design.
Second, the primers are little bit short (20mers), preferably, they should be around 25mers.
Third, the primers do not provide discrimination against unmodified DNA, ie, they do not contain non CpG 'C's in their sequence.
I think a primer re-design is necessary.
Good luck.