Primer selection guidelines
Featured picture from here.
Standard primer sequences
Outlined below are the primer sequences that we have used through the years in our STANDARD reactions. You may want or need to have these custom primers synthesized. They may differ a little from proprietary primers. That does not mean that primers purchased from conventional vendors will not work - we have simply found that this set of primers works well when sequencing with a wide array of vectors. You can order these primers or custom primers via the Keck Biotechnology Resource Lab.
Primer | Sequence (5'-3') |
M13-21 | TGT AAA ACG ACG GCC AGT |
M13-47 | CGC CAG GGT TTT CCC AGT CAC GAC |
M13-REV4 | TCA CAC AGG AAA CAG CTA TGA C |
T7 | TAA TAC GAC TCA CTA TAG GG |
T7TERM | GCT AGT TAT TGC TCA GCG G |
T3 | ATT AAC CCT CAC TAA AGG GA |
PCRIIT7 | CGA CTC ACT ATA GGG CGA ATT GGG |
PCRIISP6 | GGT GAC ACT ATA GAA TAC TCA AGC |
SP6 | GAT TTA GGT GAC ACT ATA G |
poly TG | (T)25G |
poly TC | (T)25C |
poly TA | (T)25A |
SK | TCT AGA ACT AGT GGA TC |
KS | CGA GGT CGA CGG TAT CG |
Things to note when designing primers for our services
- 20-30 nucleotides in length.
- 50% G/C content.
- G & C "clamps" on the 5' and 3' ends (at least a single G or C residue.)
- Avoid multiple Thymidine residues on 3' and 5' ends.
- Avoid primers with long runs (more than 4) of a single base.
- Avoid primers with secondary structures or that can hybridize to form dimers or hairpins. These can be easily predicted if a primer design program is used such as primer select.
- Melting temperature 55o-65oC.
- Check primers for specificity in annealing to template.
- Primers should be located at least 30-40 bases upstream of the area of interest in the sequence read.