How much GC enhancer should I add?

How much GC enhancer should I add?

For particularly difficult or high GC amplicons, the enhancer can be added to the GC Reaction Buffer to improve specificity and/or yield. The enhancer is not a buffer and should not be used alone. Final concentration of the enhancer in the amplification reaction should be between 10-20%.

When should I use Q5 GC enhancer?

The Q5 High GC Enhancer is not a buffer and should not be used alone. It should be added only to reactions with the Q5 Reaction Buffer when other conditions have failed.

What is GC enhancer?

The 360 GC Enhancer is used for difficult-to-amplify templates, especially for templates with high GC content or GC-repeats. The enhancer can also increase specificity in reactions that generate non-specific products for those templates which have a localized GC-rich sequence.

Is Q5 Hot Start?

Q5® Hot Start High-Fidelity DNA Polymerase is a high-fidelity, thermostable, hot start DNA polymerase with 3´→ 5´ exonuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. The addition of an aptamer-based inhibitor allows room temperature reaction setup.

What is in Q5 buffer?

The 5X Q5 Reaction Buffer contains 2 mM MgCl2 at final (1X) reaction concentrations and is recommended for most routine applications. For GC-rich targets (≥ 65% GC), amplification can be improved by the addition of the 5X Q5 High GC Enhancer.

Why DMSO is used in PCR?

DMSO binds to the DNA and prevents the reannealing of single-stranded DNA. It also facilitates the annealing of primer with a templet. Therefore, it increases the specificity and yield of the PCR reaction. Generally, the GC content of the template DNA for PCR is between 45% to 52%.

How much DNA do I add to PCR?

In a typical 50 µL reaction, 1–2 units of DNA polymerase are sufficient for amplification of target DNA. However, it may be necessary to adjust the enzyme amounts with difficult templates. For example, when inhibitors are present in the DNA sample, increasing the amount of DNA polymerase may improve PCR yields.

What is a hot start enzyme?

Hot Start PCR is a technique that reduces non-specific amplification and offers the convenience of reaction set up at room temperature. At permissive reaction temperatures reached during PCR cycling, the polymerase dissociates from its inhibitor and commences polymerization.

Why does DNA polymerase have high fidelity?

Replication fidelity. Replicative polymerases achieve high fidelity of DNA replication by employing several mechanisms: (1) sensing proper geometry of correct base pair, (2) slowing down catalysis in case of a mismatch, and (3) partitioning the mismatched primer to exonuclease active site.

Which does a successful PCR require?

PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest. In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced.