Labs across the world spend a great deal of money on commercially manufactured Taq polymerase for PCR every year. But you don’t have to: Many labs choose to save money by making their own Taq (and Pfu for that matter) for routine applications.
This post is not intended to provide a step-by-step protocol for making your own Taq polymerase (although links to such protocols are provided below). Instead, I want to suggest that you might want to think about doing so to save your lab some money. I’ll also provide some general tips accumulated from years of experience and various sources that might make the process a bit easier if you decide to do so.
Now, obviously, you can’t make the polymerases you’ll need for every PCR-based application. Many techniques, such as PCR-based cloning and site-directed mutagenesis, require high fidelity or highly processive polymerases that are still covered under active patents and are not widely available. However, if your lab does a lot of genotyping or other PCR-based techniques that do not require either high fidelity or processivity, homemade Taq may be the way to go.
Not only is it inexpensive to make (almost like ‘free PCR’), but it is also easy to make lots of it: A single, relatively simple, prep can easily generate hundreds of thousands of units of highly purified polymerase per liter of culture. Put another way, one batch can provide a lab of 10-12 people with enough enzyme to last for years (if it is stored properly).
Tips and suggestions for making home-made Taq polymerase
- You’ll need a plasmid then. Don’t have a Taq expression plasmid? No problem. Academic researchers can acquire bacterial expression plasmids containing Thermus aquaticus (Taq) polymerase here, and <i>Pyrococcus furious</i> (Pfu) polymerase here.
- Getting a high yield. Transform your plasmid into bacteria just before your purification, rather than relying on glycerol stocks; this will significantly increase your yield.
- Keeping it stable. Adding NP-40 to your purification solution appears to improve enzyme stability.
- (Very) easy removal of unwanted proteins. Remove bacterial protein contamination with a simple heat treatment (75°C) – most bacterial proteins will denature and precipitate, while Taq (due to its high thermostability) will remain soluble.
- Be careful with the precipitation. Titrate the amount of polyethyleneimine (PEI) used to precipitate the enzyme, as the required concentration is variable, and adding too much will inhibit precipitation.
- Adjust the final concentration. Similarly, always titrate the final enzyme concentration. Too much enzyme will interfere with your downstream PCR applications.
- Store it properly. After dialysis, store the purified enzyme in 50% glycerol (in 50–100 µl aliquots) in the -80°C – it’s stable for years. Working stocks can be kept at –20°C for several months.
- Combat proteases. Adding bovine serum albumin (BSA) (to a final concentration of 100 µg/mL) will help to counteract possible protease contamination in your prep, helping to stabilize the enzyme and maintain its activity.
Protocols for making your own Taq polymerase
- Engelke et al., (1990) Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal Biochem. 191(2):396–400. An early paper describing the cloning of Taq, including purification details.
- Farralli et al., (2007) Making Taq DNA Polymerase in the Undergraduate Biology Laboratory. Bios 78(2):69–74. Describes an adapted Taq purification for use in undergraduate teaching labs. Also good if you just wanted to make smaller preps for your own lab.
- Moazen et al., (2012) Optimization of Taq DNA polymerase enzyme expression in Escherichia coli. Adv Biomed Res. 1:82. Recent report on improved Taq purification.
- pTAQ Plasmid Information. Web based resource with many details about Taq, including a full purification protocol.
- [Bio101] Preparation of Taq DNA Polymerase. Another web based Taq purification protocol.
For more tips, tricks, and hacks for getting your experiments done, check out the Bitesize Bio DIY in the Lab Hub.
Originally published 27 March 2013. Updated and republished 12 March 2015