Please follow these guidelines when submitting samples for sequencing:
Submit templates and custom sequencing primers at the following concentrations:
| Single Stranded Plasmid | 5-25ng/ul |
| Double Stranded Plasmid | 25-150ng/ul |
| PCR fragments | |
| 100-200bp | 1-2ng/ul |
| 200-500bp | .25-1.5ng/ul |
| 500-1000bp | 1-10ng/ul |
| 1000-2000bp | 2.5-20ng/ul |
| >2000bp | 5-25ng/ul |
| BAC / Cosmid Templates | 100-500ng/ul |
| Sequencing Primers | 3ul of 3uM |
All templates and custom primers must be combined into one tube per sequencing reaction. Samples should be submitted in numerically labeled strip tubes with strip caps (we recommend strip tubes from Genesee Scientific [1], P/N 22-705.), or 96-well plates for samples of 80 or more. The online submission form can contain detailed template and primer names. A minimum of 5ul of template and 3ul of primer at 3uM are needed per sequencing reaction. This allows us to repeat the reaction if needed. The T7(s), T3, SP6, M13 (-21) Forward, and the M13 reverse primers [2] are provided by the Seattle BioMed sequencing facility at no additional cost. Please indicate in the comments section of the online request form if you need house primers added.
All DNA and primers should be resuspended in water or Tris buffer. Custom primers should be 18mer or larger with roughly a 50% GC content. Melting temperatures (Tm’s) should be around 50 degrees Celsius.
Combined template and primer should be submitted in PCR strip tubes with strip caps. Tubes with individual or attached caps will not be accepted. We recommend strip tubes from Genesee Scientific [1], P/N 22-705.