DNA From PCR Products
In case you are sending DNA from PCR products, it is EXTREMELY IMPORTANT that the DNA template is clean and free of contaminants. Please purify PCR products using the Qiaquick PCR, gel extraction kit (QIAGEN®) and PureLink purification PCR kit from Invitrogen. We recommend that an agarose gel is run after the PCR cleanup to ensure that a single clean band is the sample that is submitted for sequencing.
Please provide 10µL of template DNA per reaction at a concentration of 2 ng/uL per 100 bases of PCR product length. For Example, to sequence a 1 kb PCR fragment, we would need at least 10µL of the purified PCR product at 20 ng/µL. We recommend that PCR product concentrations be estimated by running a small known volume of the purified PCR product on an agarose gel adjacent to a DNA Mass Ladder (such as the one supplied by Gibco BRL®).
You can quantify PCR product using a number of methods: DNA mass ladder, Qubit from Invitrogen and others.
If you do not have appropriate quantifying techniques, we can quantify the samples for you.
Our working concentration of primers is 5pmol/ul. You can send 10ul of 5pmol/ul for each reaction. Better still, you can send us your primer stock; this will be kept in our primer bank. That way, you do not have to send primer with every shipment.
Tips for Primer Design:
Please design your sequencing primers to be between 18 and 22 bases long.
Each primer should have a GC content between 50 and 60%.
It is important that there are not three or more identical contiguous bases in a given primer.
The primer annealing temperature should be between 50 and 65 degrees Celsius. Also, since sequencing through polynucleotide repeat and short tandem repeat regions is difficult, it is a good idea to design primers that will not anneal on or near these regions, because this will result in noisy sequence data.