DNA Double Helix
DNA in Pipette
Cell with DNA
ABI 3100 Genetic Analyzer
Contact: Vicki Lyle
Location:
SUNY Upstate Medical University,
University Hospital
Room 4840,
750 East Adams Street, Syracuse, New York 13210
Phone: 315 464-6544
Email: lylev@upstate.edu
Hours: Monday–Friday,
9:00 am–5:00 pm

Procedure for Submitting DNA Samples for Automated DNA Sequencing

DNA Templates From PCR Products

DNA sample purity and concentration are the two most critical factors to the success of automated sequencing reactions. It is extremely important that the DNA template be clean and free of contaminants. For purifying PCR products, we recommend either the QIAGIN QIAquick Gel Extraction Kit or Amicon Centricon 100 columns to remove unincorporated dNTPs and primers. Samples should be resuspended in sterile distilled H2O or Tris only. Do not resuspend your template in TE.

It is strongly recommended that your PCR product concentration be estimated by running a small amount of your purified PCR product on an agarose gel along with a DNA ladder. It is difficult to accurately estimate plasmid DNA and PCR product concentrations using a spectrophotometer. For direct sequencing of PCR products, it is important that the PCR reaction works well. If more than one band is present, re-PCR or low-melt gel purify. Always run a gel to determine the PCR quality and quantity.

We need 5.5 ul of template at a concentration of 2.5 ng/ul per 100 bases of PCR product length for each reaction to be run. (For example, if PCR product is 600 bases, submit 5.5 ul of template at 15 ng/ul). Always send extra template if possible, so if a reaction does not work initially it can be rerun.

DNA Templates from Plasmids

DNA sample purity and concentration are the two most critical factors to the success of automated sequencing reactions. Quality in your DNA samples is imperative to achieve good sequencing results. Your DNA template must be clean and free of contaminants. We highly recommend that customers use the QIAGEN plasmid DNA prep kits to purify their plasmid DNA samples. Samples should be resuspended in sterile distilled H2O or Tris only. Do not resuspend your template in TE.

We recommend that your plasmid DNA sample concentrations be estimated by running a small volume of the cut purified plasmid DNA (digested with a restriction enzyme that only cuts at a single site) on an agarose gel with a DNA ladder. It is difficult to accurately estimate plasmid DNA and PCR product concentrations using a spectrophotometer.

We need 5.5 ul of template for each reaction at the following concentrations:

Double Stranded Template

  • If the template is from 3–5 kb long: 250 ng/ul
  • If the template is from 5–8 kb long: 250–500 ng/ul
  • If the template is from 8–10 kb long: 500–1000 ng/ul

Single Stranded Template

  • If the template is from 3–5 kb long: 25–35 ng/ul
  • If the template is from 5–8 kb long: 50–70 ng/ul
  • If the template is from 8–10 kb long: 100–140 ng/ul

Primers

Submit primers diluted to 1 pmol/ul in distilled H2O. Do NOT dilute primers in TE. We need 1.5 ul of primer at this concentration for every reaction to be run. Always send extra primer if possible so if a reaction does not work initially it can be rerun. (Note: 1 pmol/ul = 1 um solution)

Your primers should be designed to be between 18 and 22 bases long. Primer Tm should be > 55 as determined by the formula Tm=4(G+C) + 2(A+T).

Samples can be sequenced with either our stock primers which we provide at no additional charge (M13 forward, M13 reverse, T3 Promoter, T7 Promoter, T7 Terminator, SP6 Promoter, SP6 Upstream, BGH Reverse, PGEX3', PGEX5', EGFP-C and EGFP-N), or with a custom primer which the customer would provide.