Question

DESIGN PCR PRIMERS (FWD & REV): magnesium: 1.5mM, temp. ideally 60°C, hairpin melting not above 40°C, max Delta G: not over -10, approximately 20-25 letters (?)

>GANP01013062.1_TSA:_Ixodes_ricinus_SigP-154969_mRNA_sequence
GATGTGATTTCATCTCAGGGACAGCAACCAGTCTTACCTGGCTTTCCTTCCGGTCCTACTCTCTCTCCTGGAGGACTGGG
TCGACCGTGTAGCGTCTACCGCGGTTGCGAGAGCGGCTTGTGCTGCTTATTAACAGGTCGTAGAAATCATGCTGGGGCCA
CCTGTCAACCTAAAGGGGGACCCGGAGAGCTGTGCTCAGAGGAGCAAGTCAAGGGTGGCATCTATACTACTTGCTGCCCG
TGTCTCAGAGGTCCGTGTCCACCGGGGCGGAGAGCCACGTGA

Answer 1

Forward primer: GAT GTG ATT TCA TCT CAG GGA C

Primer length: 22 bases Melting temperature :60.2^oC   Hairpin Tm: 40.2^oC Hairpin G:-1.71kCal/mol

Reverse primer: TCA CGT GGC TCT CCG C

Primer length: 16   Melting temperature :63.2^oC   Hairpin Tm: 33.8^oC Hairpin G:-0.68kCal/mol (other hairpins formed has a Tm less than this)   

The reverse primer is short in order to exclude the GC rich sequence that is increasing the overall Tm of the primer.

the primers are designed using IDT oligoanalyzer