Primers:
The UCDNA Sequencing Facility provides the following universal primers free of charge: Take a look at our new universal primers!!
Primer | Sequence |
---|---|
M13F(-21) |
5' GTAAAACGACGGCCAGT 3' |
M13R |
5' CAGGAAACAGCTATGAC 3' |
T7prom |
5' TAATACGACTCACTATAGGG 3' |
T7term |
5' GCTAGTTATTGCTCAGCGG 3' |
T3prom |
5' ATTAACCCTCACTAAAG 3' |
SP6 |
5' GATTTAGGTGACACTATAG 3' |
CMV-F |
5' CGCAAATGGGCGGTAGGCGTG 3' |
CMV-R |
5' AGTAGGAAAGTCCCGTAAGG 3' |
BGH-R |
5' TAGAAGGCACAGTCGAGG 3' |
pBAD-F |
5' ATGCCATAGCATTTTTATCC 3' |
pBAD-R |
5' GATTTAATCTGTATCAGG 3' |
pGEX5' |
5' GGGCTGGCAAGCCACGTTTGGTG 3' |
pGEX3' |
5' CCGGGAGCTGCATGTGTCAGAGG 3' |
If you are designing your own primer for sequencing, please be sure that you use a primer design program to avoid multiple annealing sites, primer-dimer, etc. Be aware that PCR primers do not always work for sequencing since cycle sequencing is a much more stringent protocol than PCR. Please see our FAQ page for more details. Here are some quidelines when designing your own primer for sequencing:
- Primers should be at least 18 bases long to ensure good hybridization with template DNA.
- Avoid runs of identical nucleotides, especially runs of 4 or more G's.
- The G/C content should be in the range of 30-80%, preferably between 50-55%.
- For cycle sequencing reactions, primers with Tm between 50-70 degrees C produce better results than primers outside of this range.
- For primers with G/C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm high enough.
- Avoid primers that can hybridize to form dimers.
- Avoid palindromes because they can form secondary structures.
- Primers should be desalted and purified to industry standards.