How do you calculate Taq polymerase in PCR?
For enzymes “Units” means units of activity, or enough enzyme to catalyze a reaction at a certain rate. In this example, you need 1.25 Units of Taq per reaction. Solve it as a conversion: 1.25 Units(1 μl/5 Units) = 0.25 μl.
What concentration of DNA is needed for PCR?
Template DNA Nevertheless, the composition or complexity of the DNA contributes to optimal input amounts for PCR amplification. For example, 0.1–1 ng of plasmid DNA is sufficient, while 5–50 ng of gDNA may be required as a starting amount in a 50 µL PCR.
How many kDa is Taq polymerase?
Ampliqon Taq DNA Polymerase is a thermostable, recombinant DNA polymerase, which exhibits very high activity in primer extension and other molecular biology applications. The enzyme is isolated from Thermus aquaticus and has a molecular weight of approximately 94 kDa.
What is the concentration of primer used in PCR?
0.1−0.5 μM
Most PCR reactions use 0.1−0.5 μM primer. Assuming a maximum concentration of 0.5 μM and a reaction volume of 20 μL, each reaction will require 10 pmol oligonucleotide primer.
What is one unit Taq polymerase?
One unit Taq DNA Polymerase is defined as the amount of enzyme that incorporates 10 nmol of total deoxyribonucleoside triphosphates into acid precipitable DNA within 60 min at +65°C under the assay conditions stated above.
How do you dilute a Taq polymerase?
* Due to the difficulties in pipetting small volumes of enzyme, Taq DNA Polymerase can be diluted in 1X reaction buffer. For example, 1 µl of Taq DNA Polymerase is mixed with 4 µl of 1X reaction buffer and 1 µl of that mixture is added to the reaction.
What is a good 260 280 ratio for DNA?
∼1.8
The ratio of absorbance at 260 and 280 nm is used to assess DNA purity. A ratio of ∼1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower (≤1.6), it may indicate the presence of proteins, phenol, or other contaminants that absorb strongly at or near 280 nm.
What is a good concentration of DNA?
The most common purity calculation is the ratio of the absorbance at 260nm divided by the reading at 280nm. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 does not render the DNA unsuitable for any application, but lower ratios indicate more contaminants are present.
Why is Taq polymerase preferred in PCR?
Taq DNA Polymerase is highly efficient, so it becomes fully functional as it reaches its optimum temperature. It also has a half-life of more than two hours (at a temperature of 92 °C), a high-amplification capacity, and the ability to add 150 nucleotides per second.
What does Taq do in PCR?
Taq polymerase is the heat-stable (thermostable) DNA polymerase extracted from the thermophilic bacteria Thermus aquaticus. Its predominant function is in the polymerase chain reaction (PCR) technique, where it automates the repetitive step of amplifying specific DNA sequences.
What concentration should my primers be?
When designing primers, the amplicon length should be approximately 80–250 bp. A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a titration of primer concentrations between 100 and 500 nM.
How do you calculate the concentration of a primer?
The nmol yield can be used to calculate concentration for your oligo. To get a standard 100uM concentration, you must add the nmol*10 volumen (uL). For instance, if your oligo was synthesized and the nmol yield is 44.2, then you must add 442uL of nuclease-free water to get 100 uM concentration.