How to get good data
The single most important factor for success with fluorescent DNA sequencing is a clean, unique target with a single binding site for the primer. Remember, “garbage in - garbage out” (GIGO).
Additional suggestions for template purification can be found here.
We recommend rigorous purification methods: DEAE derivatized silica gel, anion exchange methods such as Qiagen mini or maxi tip kits work well. Nucleobond, BioRad Quantum and Promega Wizard SV are comparable. The product should specify that it is ideal for automated fluorescent sequencing.
For PCR products, it's essential that you confirm you have a single product that can bind the primer your using for sequencing. Check the pcr reaction products with a gel system that has sufficient resolution. The common 1% agarose gel used for checking plasmid purity does not usually have the resolution necessary to analyze PCR products. If you have more than one product, you either need to use an internal (aka "nested") sequencing primer that won't bind to the other products, or you need to gel purify the product of interest and then clean up the extracted fragment with a product such as Qiaquick, or Glassmilk.
Please contact the Core Lab (x2472).
UV Absorbance of Double-Stranded DNAAt 260 nm, 1 A260 unit is approximately 50 µg / mL. DNA from cell cultures should have a 260/280 ratio between 1.6 and 2.0 (< 1.6 indicates residual protein or phenol; > 2.0 indicates residual RNA); and a 230/260 ratio of 0.3 - 0.9 (> 0.9 indicates residual sugars).
Lots of molecules absorb UV light of 260 nm or thereabout, not just DNA. So use the absorbance at 260 nm only with DNA templates that you know are pure. I.e., no RNA, free nucleotides (sides), protein, phenol, etc.
How do you know? Run it on a gel. A 1% agarose in TE buffer with an intercalating dye like ethidium bromide or PicoGreen will give good separation for most plasmids and PCR products; adjust as appropriate for the size. Agarose gels are so simple to run, there really is no excuse to not QC your samples on a gel. Just run a similar type of DNA whose concentration you know as a control (maybe two with a low and high concentration) and interpolate (guestimate 8-) based on the intensity. If you don't overload your template, the fluorescence is fairly linear.
Dr. Lyons at U. Michigan gives a full explanation with an example.
The OHSU library has the Wiley Current Protocols on line. Here is a link to the protocol for agarose gel electrophoresis of DNA. It reads more complicated than it really is. We just keep a stock of 1% in an erlynmeyer flask, melt it when we need it, and pour enough for the number of lanes we need onto a microscope slide (6 -8 cm long). We suspend the well forming "comb" so it's in the molten gel, but not quite touching the slide. We pour a few of these at a time, and store them for a week or so wrapped in saran wrap in the frig and pull one out as needed. Tris-borate-edta buffer can be made in bulk, and has a good shelf life for this purpose. Gel rigs are cheap. You'll be happy when you know you have a clean template, it makes troubleshooting so much easier.
Hints for Gel Purification of PCR Products
If you cannot get a single, unique, PCR product and need to purify one for sequencing, there are a number of gel purification methods for purifying PCR products. Qiagen's QIAquick Gel Extraction Kit is one that has been successfully used by our clients. Here are a couple of hints specific to that kit.
- A 15 mL conical tube works best once the gel has been completely dissolved (step 3 of Qiagen protocol).
- Allow the wash solution to sit on the column (with bound DNA) for 5 minutes prior to spinning.
- Likewise, allow the elution buffer (50 µL) to sit for a minute on the column (with bound DNA) for a minute prior to spinning and collecting the eluant (with released DNA).
- A second elution (30 µL) will increase your yield.
- Amounts needed for sequencing
We recommend running your purified product on a gel and comparison to a mass ladder for quantification of your yield.
How do I sequence a PCR product directly (i.e. without cloning it)?
- PCR amplify the region of interest from your purified genomic DNA. Ideally, your PCR primers should be at least 50 nts from the region of interest. Call me if this is not the case.
- Check the product on an agarose gel (1.5% metaphor or other high quality agarose) or an appropriate non-denaturing PAGE gel.
- Purify the PCR product:
- a. IF (2) shows you have a single product, then use a simple PCR cleanup kit (Qiagen, Promega, Sigma, others) to remove primers and unincorporated dNTPs. Go to 4a.
- b. BUT IF (2) shows more than one product from your PCR reaction, then use use the simple cleanup kit and go to 4b. OR make the PCR conditions more stringent until you get a single, unique product. Then clean it up (3a) and sequence as in 4a OR try PCR Rescue: Making one band from many The bitesizebio.com PCR-Rescue method for gel separation of multiple products, capture the fragment of interest, and reamplify.
- Sequence the purified PCR product
- a. directly with one (and in a separate sample use the other from the opposite direction) of the PCR primers.
- b. Use a nested (aka internal) sequencing primer with the purified PCR product. This, in theory, should only bind to the region of interest.