For plasmids: From our experience, it is CRITICALLY important that the DNA template be clean and free of contaminants! The success of your sequencing reaction depends on many things, most importantly on the purity and cleanliness of your DNA. We recommend Qiagen, BioRad, and Clonetech kits for plasmid purification. Most importantly, whichever prep you choose, do not resuspend in TE. The EDTA in the TE acts as a chelating agent capturing the Mg2+ needed as a co-factor for the polymerase in our cycle sequencing reaction. This will inhibit your reaction and could cause it to fail completely. We recommend resuspending your DNA in ddH20. Qiagen EB buffer or Tris 8.0 are also fine.
For PCR products: The DNA should be purified using a reputable PCR purification method such as QIAquickTM or AMPureTM to remove excess primers and dNTP’s after your PCR cycling. Please run your PCR products out on a gel to be sure that you do not have multiple products. Elute your PCR products with ddH20 or Tris 8.0 buffer, no TE here either.
DNA Concentration Guidelines
A word about DNA concentrations...we recommend that you not only UV spec your samples, but also double check them on a gel to check concentration.
Here is what we need from you....
Template DNA: We need 8µL of template DNA per reaction placed in a 0.5mL tube. Cycle sequencing reactions only use one primer in each reaction, so if you are running one forward and one reverse primer to confirm sequence, we will need a total of 16µL of template DNA.
Please be sure the names on the tops of your tubes correspond to what is on your request form.
Primers: Please supply us with 6µL of your own primer per reaction at 3µM, or 20ng/µL, unless you are using our facility supplied universal primers. Again please be sure the names on the tops of your tubes correspond to what is on your request form.
For larger orders please note: If you will be taking advantage of our bulk discount pricing and submitting 48 or more samples at once, please submit your samples in a v-bottom 96-well PCR plate (click here for diagram). Please aliquot your samples according to our directions. Samples 1-8 should be in wells A1-H1, samples 9-16 should be in wells A2-H2, etc. For your convenience, you will notice that these well numbers correspond to the well numbers on our request forms. Be sure to label plate and primer/s clearly with your name and order number and cover the plate with an adhesive seal (plastic or foil). Please do not use strip caps as contamination can be a problem.