Critical to the success of your sequencing is purifying templates properly. Poor template quality is the most common cause of sequencing problems such as noisy data (peaks under peaks), failed sequence and/or overall weak signal. The method of purification affects the success of Sanger Sequencing Products. Purification reduces contaminants including: excess PCR primers, dNTPs, enzymes, and buffer components; Proteins, RNA, Chromosomal DNA, residual salts, residual organic chemicals such as phenol, chloroform, ethanol, and residual detergents.
PCR Purification Methods:
The protocol we’ve used for many years is easy and requires the addition of a few reagents to PCR products, followed by a quick incubation in a thermal cycler. It is highly recommended that each unpurified PCR product be visualized on an agarose gel to determine the quality and quantity prior to treatment. Don’t waste your precious grant funds on expensive columns or pre-mixed solutions, make your own cocktail as described below.
Antarctic Phosphatase (NEB* cat#M0289S/L)
Exonuclease I (NEB* cat#M0293S/L)
0.5uL Antarctic Phosphatase Buffer
0.6uL Antarctic Phosphatase
0.6uL Exonuclease I
4.0uL Unpurified PCR Product (this is a starting point, more or less template could be needed depending on concentration)
Thermal Cycler Protocol:
37 degrees C for 20 minutes
80 degrees C for 20 minutes
After incubation, add 1μL of 5μM (micromolar) primer NOTE: 5 μM is equal to 5 pmol/μL for those who wish to work with pmole amounts.