To produce a successful sequencing reaction templates must be purified and quantified 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. Before purification it’s imperative that each unpurified PCR product should be visualized on an agarose gel to determine the quality and quantity prior to treatment.
PCR Cleanup Methods
The method of purification affects the success of Sanger sequencing. Purification is essential to remove unincorporated primers, left-over dNTPs, enzymes, buffer components, proteins, RNA, Chromosomal DNA, residual salts, residual organic chemicals such as phenol, chloroform, ethanol, and residual detergents- basically anything that can interfere with the cycle sequencing reaction. For each method there are benefits and trade off’s and your choice will depend on your budget, available equipment and consumables, and know how
Columns are fail proof
Column kits are available from companies such as Qiagen and Zymo, shop arond and you’ll find kits for as low as $.50 per sample. The benefit to using these kits is they are generally fail proof and require equipment readily available in most molecular labs.
PEG precipitation is inexpensive but time consuming
This costs just pennies per sample but requires a time commitment and spinning plates (for an eternity). Skilled hands are necessary as well to not disturb the precipitate. Polyethylene Glycol (PEG) recipes and protocols are readily available online. I like this one from Travis Glenn.
Enzyme purification is easy and won’t drain the budget
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. Buy a ready made mixture, ExoSAP-IT or make your own by searching for recipes and protocols online. A few points to consider, A) Shrimp Alkaline is better then other enzymes, B) most protocols will recommend using 4ul of PCR, but this is a starting point, C) lastly, we recommend extending the inactivation time to 30 minutes to make sure the enzymes are completely deactivated. The trade off for this method is the sequencing quality can be compromised because of the salts and buffers remaining in the reaction. I suggest testing the method before making a huge investment in reagents. This method works well for some templates and not at all for others.
SPRI (Magnetic) Beads require and initial investment of ~$1,400, but lasts forever
Solid Phase Reversible Immobilization beads can be purchased from companies like Agencourt and Kapa, but are exorbitantly expensive. Make your own following this recipe and save a ridiculous amount of money. Prepare yourself for an initial investment of about $1,400. This includes $600 for the reagents (mostly the Sera-Mag SpeedBeads) and another $800 for a 96-well plate magnet stand.
Most labs labs will want to own 2 types of magnet racks but for PCR purification you’ll only need the DynaMag -96 Side Magnet Cat #12331D. Beware not all magnets are created equal. I’ve tested many of them and strongly recommend the side binding rack because of the 13th row, which allows 12 row plates to be moved back and forth to expose the magnet pellet to ethanol during the wash steps.