First I want to thank you all for this great forum and the help I already get from here. But right now Iam a little bit lost and hope I can get specific help from you all. Iam only a diplomastudent get the big task to establish direct bisulfide sequencing in our lab and nobody is on hand to really help me.
Okay for example this is the region of interest (not converted):
AATACTGGAAGGACAGCGCCTGACTGGCTTTTAAACACTCAGTTGTGGAATACATCCCAC GCTTTTGTGAACTCCTTTGTTTCCCTGACAGACCCGCCCCCAGGACTCCACCCTCACTCT GCCGGCAACACAGTTCACACACTGCGGCCTGCCACACCCACCCCATCACTCAGGAGGAAC AAAAGCCAATCACAGGGTGAAAGGAGTCTTTGGTATCAGGCAGCGAGCACAGTATTCTTG GAGTGTGCGAGGCAGGGCTAGGCTCAGAGGTCCTTCTACCACTAAATGCTGCTACTATAG CAGAGGAACAAGCATAGTCAATAAAGACAAAGGCAAGTCTAAGATGCTCCAAAGTGTCCA AAGTTATGATCTTTTGGATTTTTCTTTTTTCCAATTACACTCGCTTTCGTAGGATCACTA GTCTTTTCCTAAAGTTGTATGTAATGGGCGTGTGCGCCTGGACAGACATTGACCTCCAGG GATGAAGAATGAGGCACTACTCTCGCCAAGTGCTTTGGCTTGGCGAAGCCTGCTCAGAGT AAGAAGAGGAATTGCTCAAGTAACGCAAATGACCCTAAAGCTCCCTACCAGTGCTCACTC GCAACACCGGACTGCACCTGCTGCACCCACCCATTAGCAGCCCCTTCTCCATCCCCAGCC TATGGTACTAAATCACCCCGCCCCGCCACACTACCGCAGTCTTCACGCTCCCAGTTCCCA GTTCCCCACACTCAGCTTGCCCTTCTCAAGTCCGAGACAATGCTCAGGCCCCAGCTTCTC CCAAGAAGCCCCACTCCTCGCAAGGTCTTCACGCCCGCGCTGATTGGCCACAAGCCCATA GTCCAGCCCCCAAGACCGTCCCTGGGGCTCAGAGCATTACGTCAACCCGGCCTGGAGAGC GCTAGCGAGAACGGGACTGGGGGCTTTGGCTGGGGGGCGCGGTCTTTTTGGTTCGCCCCA CCCTCCCTACATAAAAGACCGAACCCACCCGGCCCTCGCCCTCAGCCCGTAGCCCGTCGG TTCCGGAGTAAGTTCCAGGTGGCCCAGCAGTGGGTGTGGAAGGGGAGGATCATCAGACCC ACTGACACAGACCCAAGACAGCAAGAAGCTAACCAGGCACCATGCGAGAGATCGTGCACA TTCAGGCGGGCCAGTGCGGCAACCAGATCGGTGCCAAGGTGGGTTCACTGCTTAGGGGGT GAGGGAGGGACCCTAACGGAGCTCCCCTCGGGATAGGTGGGGTCCAAAATGAGGCGCGGC
I want to amplify the region between 800 and 1000 bp (use Methprime options:
Exclude Regions: 1, 750 1100, 120
Max. poly T: 3
As forward primer I design TTGATTGGTTATAAGTTTATAGTTTAG (len. 27 bp, Tm 55.12 )
As revers primer I design CTTCCACACCCACTACTAAAC (len. 21 bp, Tm 57.63)
Tm I calculate with http://www.sigma-gen...alc/DNACalc.asp because we want to order the primer from that company.
Are the primers alright so far?
To make it easier for our sequencing facility I want to use M13 Tails for the Primer. So i can also use a touchdown PCR to amplify the product.
Okay and that is the big question. If I add the M13 Tail to the primer (TGT AAA ACG ACG GCC AGT) I get really high Tm (over 70 degree ) and also extreme long Primer (about 40 bp). What should I do, cut the gen specific part of the primer to get smaller primer (but also more unspecific) or can I work which these long primers with the high Tm ? Sorry for that dumb question but as I sad Iam pretty new in these field.
Big thanks for help!
The M13-tail is at the 5'-end of one of the primers and is not annealing to the sample DNA (but later to the M13-sequence that is incorporated to the amplified DNA). Therefore you don't need to consider it in the calculations.
The experiments I did and also from several others I know about, used the Schuelke (2000) paper and followed all more or less his protocol and settings and did surprisingly few modifications (especially primer concentrations and lowering Ta after a the usual cycles). So I'd follow his suggestions too first.