Frequently Asked Questions

A: Klentaq is a truncated version of Taq, which lacks the 5’>3’-exonuclease domain of the enzyme. Klentaq has improved fidelity and is more thermostable and robust than Taq. The fidelity of Omni Klentaq vs. Klentaq is not compromised. Cesium Klentaq has slightly better fidelity than Klentaq. The fidelity of the rest of the mutant enzymes has not been fully determined yet.

The sensitivity of our mutant enzymes is not compromised at the expense of their special inhibition-resistance or cold-sensitivity features, when compared to top commercial Taqs, and allow single gene copy detection from human DNA. The Klentaq enzymes typically require longer than Taq extension times for high yield amplification, and we recommend 3-4 minute extension per 1 kb DNA target.

A: The “LA” stands for “long and accurate”. The LA enzymes are blended with a proofreader, and perform with better fidelity.  Also, they are the enzymes of choice for long DNA targets, with which they perform exceptionally well. In addition, they are generally more robust, therefore short targets amplification can also benefit from them.

*TIPS on long targets:  For purified DNA templates, consider enzyme concentration of at least 0.1 ul/1 kb target/ 50-100 ul reaction volume, and allow 3-4 min extension time per 1 kb target.  For crude samples, more enzyme may be needed, depending on the level of inhibition (enzyme titration is strongly recommended). Consider including our PEC enhancers.

A: Our novel enzymes are mutants of Taq DNA polymerase, specially selected for their high resistance to widely encountered potent PCR inhibitors, such as whole blood, serum, plasma, urine, humic acid, bile salts, plant tissues extracts, various food related inhibitors, GITC, ethanol  and others.  As a result, these enzymes can tolerate such inhibitors in PCR at levels where most commercial Taq products fail to perform. This unique feature of the Taq mutants allow for direct PCR of various clinical, environmental, and forensic crude samples, skipping the DNA extraction steps.  Our PCR Enhancer Cocktails (PECs), specially formulated for most inhibitory crude PCR samples, can further raise the tolerance of reaction to inhibitors. In addition, some of our PECs are designed to help also with difficult, GC-rich targets. Due to the above mentioned qualities, our novel PCR enzymes and enhancers should certainly reduce the labor time, cost, and false negative results of your challenging PCR.

A: Our unique selection of mutant Taq enzymes, rendered tolerant to various potent PCR inhibitors, along with our PCR enhancers, enables a variety of direct PCR applications, skipping in many cases DNA extraction prior to PCR. The choice of enzyme / enhancer depends on the type and concentration of the inhibitory substance, the type of qPCR assay, hot-start PCR dependence, GC-content of the template, and other factors. Please refer to Tables “Which Enzyme is Best for Me” and “Which Enhancer is Best for Me”.

A: All of our enzymes work quite well with intercalation dyes, like SYBR Green.  Actually, they possess 2-3 times higher than wild-type Taq resistance to SYBR Green (this dye is inhibitory to PCR for plain Taq at more than 1X concentration), which gives them certain advantage in qPCR, especially in reactions with some fluorescent quenching.

For TaqMan assay we only recommend our full-length Taq mutants (the OmniTaq series or CesiumTaq), but not the Klentaq ones, as they lack the 5’>3’- exonuclease activity, required for cleaving the TaqMan probe.

Tip: For tough / inhibitory qPCR samples, 1:1 blends of full-length Taq and Klentaq enzymes may work better than the full-length enzyme alone. (The Klentaqs are generally more robust, and a half amount of the full-length Taq enzyme is sufficient for probe cleavage). If you would like to try this, the two buffers should be blended in the same proportion as well.

Note: The LA versions of our enzymes are not optimal for TaqMan assay, due to the proofreader activity in them.

A: We suggest that you select Omni Klentaq, Omni Klentaq 2, OmniTaq or OmniTaq 3, combined, if necessary, with PEC. We strongly recommend enzyme titration for such crude samples, as more enzyme helps with more blood.  Please keep in mind that the optimal amount of enzyme for samples with blood would probably be far in excess of the optimal amount of enzyme for purified DNA samples, an excess enzyme may result in over amplification. Usually 5-10 times less enzyme is recommended with purified DNA.  If even higher amount of the enzyme (at least 1 ul/25 ul reaction) is not satisfactory, you can include PEC enhancer to further boost the blood resistance. We do not recommend using PEC for your purified DNA unless the target is GC-rich and tough. 

You may also use our 5X PCR kits formulated with these enzymes (which provides convenience, but limits the option to titrate the enzyme).

As per the cycling conditions, we suggest that you use your target-optimized PCR protocol with two changes:  A. Introduce a longer initial heating step of 8-10 min at 94-95 deg. prior to cycling, and B. double or triple your extension time.  Please note: If you use a PEC enhancer, keep in mind that it may reduce your optimal annealing temperature by several degrees.

Note: The same considerations apply for other inhibitory crude samples after selecting the right enzyme for your application.

A: Yes, with some easy changes in the protocol. Our novel enzymes can resist as high as 40% blood in PCR. Therefore, in qPCR of blood, the amplification should be OK (and you should see your expected products if you want to run them in agarose gel). However, a complication with the optical detection of the amplified products in qPCR stems from the fluorescence quenching effect of the heme. If you use SYBR Green, a remedy to this problem is to simply raise the input dye concentration to 20-30 X (for 5-10% blood), or even higher, depending of the blood concentration. This will compensate for the quenching effect, and will reduce the background.

If you use TaqMan assay, again due to the quenching effect of blood, we recommend raising the fluorescent probe concentration to 400-500 nM, and not exceeding 4-5% blood.

Note: these protocol changes are not necessary if you amplify serum, plasma, or other non-quenching crude samples.

A: Some DNA extraction kits cannot completely remove PCR inhibitors from the starting material, such as blood or plant tissue components or humic acids, and can even introduce some inhibitors, such as traces of phenol, chlorophorm,  guanidinium thiocyanate or ethanol. This problem may be eliminated by using our inhibitor-resistant mutants of Taq after a careful titration of the enzyme. (This should be a relatively “easy job” for our enzymes as compared to crude PCR samples, and you’ll have to make sure you don’t overdose the enzyme).

A: Our cold-sensitive enzymes, such as Cesium Taq, Cesium Klentaq AC, and Cesium Klentaq C, display a suppressed activity at low temperatures, while they become fully active above 65 degrees, thus performing with an intrinsic hot-start in PCR. Unlike hot-start Taq formulations, involving anti-Taq antibodies or chemical modification of the enzyme, they do not require any changes in the cycling conditions.

A: The enzyme Units concentration provided by some Taq manufacturers is determined by a slightly modified version of the conventional DNA polymerase activity assay, developed earlier for non-thermostable DNA polymerases. This assay, based on a relatively simple and “easy” reaction, only measures the nucleotide incorporation into nicked DNA at 72 degrees, and does not account for important enzyme qualities in PCR conditions, such as thermostability and processivity. Thus, it is not a per se PCR assay, and does not deliver actual “PCR units”. As a result, it may not manifest and predict the real enzyme performance in PCR. Therefore, we, like other manufacturers, believe that providing a recommended amount of enzyme per reaction is more reliable and practical for the final user. Our suggested enzyme volumes per reaction are based on a series of enzyme titration assays with various target types and sizes.

A: We ship our enzymes all around the world from our labs in the US.  Most orders ship with Fed Ex and arrive domestically within one business day of placing the order and internationally within one week of placing the order.