DNA Sample Preparation For Automated Sequencing

DNA From PCR Products

From our experience, it is CRITICALLY IMPORTANT that the DNA template be clean and free of contaminants. Please submit samples resuspended in STERILE ddH2O or Tris and NOT TE. For purifying PCR products, we recommend using either the QIAquick Gel Extraction Kit (supplied by QIAGEN®) or Centricon® 100 columns (supplied by Amicon®) to remove unincorporated dNTPs and primers. Please provide us with 8µL 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 8µL 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®).

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.

DNA From Plasmids

From our experience, it is CRITICALLY IMPORTANT that the DNA template be clean and free of contaminants. Please submit samples resuspended in STERILE ddH2O or Tris and NOT TE. 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 8µL 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*

We have found that DNA samples greater than 10 kb yield inconsistent sequencing results compared to small plasmids. If you have a BAC, cosmid or phage to sequence, please contact us first for our modified concentration recommendations.

Other Items Of Importance

Primers: Please provide primers at a concentration of 3µM or 20 ng/µL or 3 pmol/µL. We need 8µL per reaction. This is enough to do the reaction twice if needed.

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 percent . 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.

Davis Sequencing Supplied Primers:

The following primers will be provided for FREE by our facility;
SP6 promoter primer: 5'-TAT TTA GGT GAC ACT ATA G-3'
T3 promoter primer: 5'-ATT AAC CCT CAC TAA AGG GA-3'
T7 promoter primer: 5'-TAA TAC GAC TCA CTA TAG GG-3'
M13 R primer: 5'-TCA CAC AGG AAA CAG CTA TGA C-3'
M13 (-21) primer: 5'-TGT AAA ACG ACG GCC AGT-3'
T7 terminator primer: 5'-GCT AGT TAT TGC TCA GCG G-3'
BGH REV primer: 5'-AAG GCA CAG TCG AGG-3'
CMV FOR primer: 5'-CGC AAA TGG GCG GTA GGC GTG-3'
pBAD FOR primer: 5'-CCA TAG CAT TTT TAT CCA TAA G-3'
pBAD REV primer: 5'-GAT TTA ATC TGT ATC AGG CTG-3'
pGEX5 primer: 5'-GGG CTG GCA AGC CAC GTT TGG TG-3'
pGEX3 primer: 5'-CCG GGA GCT GCA TGT GTC AGA GG-3'

DNA samples and primers cannot be stored once the sequencing reaction is successfully performed due to freezer space limitations.
Please do not submit DNA samples or primers that you would like returned to you at a later date.