PCR Product Protocol

When bringing a PCR product to the DNA Analysis Facility for automated sequencing, our recommended procedure is as follow:

1. Ethanol precipitate the product of a 50 or 100µl PCR reaction.
2. Resuspend the precipitate in approximately 10µl of loading buffer.
3. Load into one lane of a pure Nusieve gel (2-3%). (Please do not use agarose as it may contain impurities which affect the sequencing reaction.)
4. Take a picture of the gel.
5. Cut out the appropriate band(s).
6. Extract the DNA using the Microcon/Millipure kit from Amicon, or a similar purification kit.

To obtain optimal sequence, you should have an isolated band that is free of primer-dimers. Please quantitate PCR products by using a DNA dipstick from Invitrogen (available at the CORE store). We do not recommend that you use a spectrophotometer to O.D. PCR products, because you will not get an accurate reading. Quantitation of products by a fluorometer is acceptable, but the dipsticks are easier and provide a permanent record of the concentration. If you are quantitating via standards on a gel, please use a standard curve to increase accuracy.

We need the template to be at 3ng/µl/100bp of template. All templates and primers must be in water and at appropriate concentrations. We will not quantitate your product for you.