DNA From Plasmids
DNA From Plasmids From our experience, it is EXTREMELY IMPORTANT that the DNA template is clean and free of contaminants. Please submit samples resuspended in STERILE ddH2O. QIAGEN® and Bio-Rad Quantum® minipreps are two of the most consistent methods for plasmid DNA preparation. Some plasmid DNA preparation methods are not compatible with automated sequencing, so please contact us if you have any questions. We recommend that plasmid DNA sample concentrations be estimated by running a small known volume of the cut purified plasmid DNA (digested with a restriction enzyme that only cuts at a single site) on an agarose gel adjacent to a DNA Marker with known DNA band amounts.
Since it is difficult to accurately estimate plasmid DNA and PCR product concentrations using a spectrophotometer, we do not recommend using a spectrophotometer to estimate your template DNA concentrations.
Listed below are our recommended DNA concentrations for a given size plasmid sample (vector DNA + insert DNA).
We need 10µL per reaction at the following concentrations:
Double Stranded Template
If the template is from 3-5 kb long:100-200 ng/µL If the template is from 5-8 kb long: 200-400 ng/µL If the template is from 8-10 kb long: 400-500 ng/µL*
Single Stranded Template
If the template is from 3-5 kb long: 25-35 ng/µL If the template is from 5-8 kb long: 50-70 ng/µL If the template is from 8-10 kb long: 100-140 ng/µL*
Primers: Please provide primers at a concentration of 3 pmol/µL. We need 10µL per reaction. This is enough to do the reaction twice if needed. Also, you can send your stock of primers to the lab. This can be stored in our primer bank and used each time samples are sent for sequencing.
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.