PRIMER DESIGN
The Vanderbilt University DNA Sequencing Core Facility offers the following stock primers:
|
Primer Name |
Primer Sequence |
|
T7 |
5’ TAA TAC GAC TCA CTA TAG GG 3’ |
|
T3 |
5’ ATT AAC CCT CAC TAA AGG GA 3’ |
|
SP6 |
5’ GAT TTA GGT GAC ACT ATA G 3’ |
|
M13F(-40) * |
5’ GTT TTC CCA GTC ACG AC 3’ |
|
M13F(-21) |
5’ TGT AAA ACG ACG GCC AGT 3’ |
|
M13R |
5’ CAG GAA ACA GCT ATG AC 3’ |
* NOTE: If the request does not specifically ask for M13F(-21), then M13F(-40) is used.
If you are sequencing a custom-made primer, you should adhere to the following guidelines in order to ensure high quality results.
1. The Tm of your primer should be 50-58°C.
2. The GC content of your primer should be ~ 40-60%.
3. The recommended length of primers for sequencing is 17-25 bases.
4. The concentration of your primer should be ~10 ng/ul or ~1µM.
5. We highly recommend that you use primer design software such as Oligo or MacVector to check for any design flaws.
6. Do not use primers that will self-hybridize (form a primer-dimer).
7. Please submit primers in water instead of TE.