Preparation of DNA Samples & Sample Submission
The quality of the DNA template is one of the most critical factors in automated DNA sequencing. Review the steps below to maximize your chances of obtaining good quality DNA template:
- Isolating the DNA:
Plasmid preparations (either large-scale or mini-preps), numerous methods work if you are sufficiently careful to avoid genomic DNA, excess RNA, or contaminants. Many researchers use Qiagen products with good success, but other manufacturer's products may work as well. Elute in distilled water or in 1 mM Tris, if desired. Do not elute in TE, as the EDTA can interfere with sequencing. You can use Cesium Chloride preps, but make very sure there is no Cs or EDTA present in the final sample. You may need to do an extra ethanol precipitation for this reason.
PCR products, there are two options, depending on the cleanliness of the PCR reaction. If the PCR product is exceptionally pure, you can use an ultrafiltration device from either Amicon, Millipore or Qiagen that separates the PCR products from the primers, discarding the latter. Otherwise, run the PCR products out on a gel and gel-elute the desired band. Note that the PCR product must be VERY pure in order to obtain clean sequence. Extraneous bands may appear low-intensity to you, but could easily ruin the sequencing run. Note also that you MUST remove all traces of the original PCR primers, as these could produce undesired bands by acting as sequencing primers.
- Quantitation of template DNA:
If you have enough template DNA, always determine its concentration by UV absorption. However, very often, you cannot reliably quantitate a mini-prep using a spectrophotometer!
**Some comments about getting accurate spec readings on DNA:
- Readings below 0.05 AU require careful blanking in order to ensure accuracy.
- Many specs cannot be trusted below 0.05 AU, and most should not be used below about 0.02 AU.
- Remember that RNA and free nucleotides also absorb UV, so the sample must be free of these contaminants.
- Be careful to avoid chromosomal contaminant DNA. On a restriction digest, it shows up as a smear in the background. Any visible smear is likely to represent a large percentage of the total DNA present, thus throwing off your measurement.
- Be suspicious! If your yield of DNA seems unusually good, it is probably too good to be true.
If you do not have enough to measure spectrophotometrically, then run a small aliquot on a gel and estimate the amount of DNA by comparison with standards of similar size and known concentration (e.g., Low DNA Mass ladder, Invitrogen cat # 10068-013). **Actually, it is a good idea to do this even if you have performed a spec measurement, in order to cross-check yourself.
- Diluting the template to desired concentration:
After determining the concentration, dilute the template to the correct final concentration using distilled water (please no TE or other EDTA-containing buffer), and avoid adding any divalent cations (i.e. Mg, Ca, Mn).
**See “Required DNA Concentrations for Automated Sequencing”
- Submission of DNA samples:
After you have isolated, quantified, and diluted your DNA template/primers to the required concentration:
- Complete the Excel SAMPLE SHEET
Fill out the sample name and comments column only. The 'comments' column must include the name of the primer you want us to use in the cycle sequencing reaction. The primer name must match the name labeled on the primer tube. Additional information in the comments column is optional (i.e., use this column to make notations regarding your samples that may be useful for your lab when examining your sequencing results)
- Save the SAMPLE SHEET as a .txt file
- Complete the SAMPLE SUBMSSION FORM
- Email the completed SAMPLE SHEET and SAMPLE SUBMISSION FORM files to angie.ambers@unt.edu
- Drop samples off in the labeled small (2.5cu ft) white refrigerator in EESAT 325E
- Samples must be submitted in 1.5-2.0 ml microcentrifuge tubes in a rack labeled with the investigator(s) name and date of submission. Place your labeled template tubes in the rack in successive order as given in the SAMPLE SHEET, and include each of the primer(s) in the rack immediately after the specific sample(s) you want sequenced. If you have multiple subsets of samples using different primer(s), place the specified primer(s) after the particular subset of samples, followed by each successive subset of samples with their corresponding primer(s), etc... (more info). This will help improve our efficiency when organizing samples and setting up the cycle sequencing reactions.
- Template tubes must be labeled with the Sample Name and Researcher
- Primer tubes should be labeled with the Primer Name and Researcher
- Attach a paper copy of the SAMPLE SHEET and SAMPLE SUBMISSION FORM to the tube rack (i.e., place the microcentrifuge rack on top of the folded, printed forms)