What is Q5 polymerase?
Q5® High-Fidelity DNA Polymerase is a high-fidelity, thermostable DNA polymerase with 3´→ 5´ exonuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. Amplification of a variety of human genomic amplicons from low to high GC content using Q5 High-Fidelity DNA Polymerase.
What is in Q5 high GC enhancer?
Shyam deduced that Q5 reaction buffer contains glycerol, which reduces DNA secondary structure, and tetramethylammonium, which increases primer stringency; and Q5 High-GC Enhancer contains DMSO and glycerol to do more of the same.
How do you use Q5 polymerase?
Reaction Setup: All components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors. Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.
How does Q5 work?
Hello, In contrast to chemically modified or antibody-based hot start polymerases, Q5 utilizes a unique synthetic aptamer. This molecule binds to the polymerase through non-covalent interactions, blocking activity during the reaction setup.
Does Q5 produce blunt ends?
Blunt-ended products are produced by Q5 High-Fidelity DNA Polymerase (and all Q5 product formulations). If needed for TA cloning , 3´A-overhangs can be added with a different polymerase. It is very important to remove all the Q5 High-Fidelity DNA Polymerase first by purifying the PCR product.
What is high-fidelity polymerase?
The fidelity of a polymerase refers to its ability to insert the correct base during PCR. High-fidelity PCR, utilizes a DNA polymerase with a low error rate and results in a high degree of accuracy in the replication of the DNA of interest.
What does Betaine do in PCR?
Betaine is the most common PCR additive used to enhance amplification of GC rich sequences because of its ability to dissolve secondary structure that blocks polymerase action.
How much DMSO do you use in PCR?
Generally, the GC content of the template DNA for PCR is between 45% to 52%. If the GC content is higher than the desired range use 5% DMSO in PCR reaction. 4% to 10% DMSO concentration can be utilized to optimize the PCR reaction.
Does Q5 leave an overhang?
The proofreading activity of Q5 is very strong, so any residual polymerase will degrade the A-overhangs as they are added. Taq DNA Polymerase or Klenow (exo-) DNA Polymerase are excellent options for A-overhang addition. We recommend ligating immediately so the 3´A-overhangs will not be lost during storage.
Do PCR products have 5 phosphate?
As mentioned above, your PCR products don’t have 5’Phosphate. Two choices – you could order Phosphorylated primer or you could use PNK to put phosphate at the end (use some PEG( (10-20%)) to improve on phosphorylation).