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ONE OR MORE PRIMER SEQUENCES

1. Go to the Primer BLAST submission form.
2. Enter one or both primer sequences in the Primer Parameters section of the form.
3. In the Primer Pair Specificity Checking Parameters section, select the appropriate source Organism and the smallest Database that is likely to contain the target sequence.

What makes a good primer?

1. Aim for the GC content to be between 40 and 60% with the 3′ of a primer ending in G or C to promote binding.
2. A good length for PCR primers is generally around 18-30 bases.
3. Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other.

If you know the positions of each primer with respect to the template, the product is calculated as: Product length = (Position of antisense primer-Position of sense primer) + 1. 2. Product Position: Primer can be located near the 5′ end, the 3′ end or any where within specified length.

How can I tell if I have primer-dimers in my PCR reaction? In quantitative (real-time) PCR, primer-dimers will appear as a peak with a Tm lower than the Tm of the specific product. This peak will also appear in the no-template control (NTC).

1. increase the annealing temperature.
2. increase time/ temperature of template denaturation.
3. decrease primers concentration(10 pmol will be OK)
4. use a PCR enhancer such as DMSO.
5. Check out your template.
6. use high quality Tag.

Primer-dimer is when the PCR product obtained is the result of amplification of the primers themselves. This sets up a competitive annealing situation between the template and the primer-dimer product during amplification, negatively affecting results downstream.

Self-complementarity is the likelihood that the primer will bind to itself and to the other primer in the pair. Low self 3′-complementarity score: Pick primers which have a low self 3′-complementarity score, as given in the NCBI Primer-BLAST detailed primer report.

A primer is a short, single-stranded DNA sequence used in the polymerase chain reaction (PCR) technique. In the PCR method, a pair of primers is used to hybridize with the sample DNA and define the region of the DNA that will be amplified.

Like other DNA polymerases, Taq polymerase can only make DNA if it’s given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis. Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied).

Every primer is a brief piece of RNA complementary to the original DNA strand. DNA polymerase can’t copy the DNA without a primer. In short, when DNA needs to be copied, RNA primers act as a DNA polymerase starting site. Option D is incorrect.

The various components required for PCR include a DNA sample, DNA primers, free nucleotides called ddNTPs, and DNA polymerase. The various components required for PCR include a DNA sample, DNA primers, free nucleotides called ddNTPs, and DNA polymerase.

There are several things that may improve yields:

1. Check the primer design using computer software.
2. Optimize the annealing temperature in a 1-2°C step.
3. A primer concentration of 0.2 μM is satisfactory for most PCR reactions.
4. Increase cycling numbers up to 45 cycles.
5. Do a manual hot-start.
6. Use thin-wall 0.2 ml PCR tubes.

To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA.

Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA. Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

Self-complementarity is the likelihood that the primer will bind to itself and to the other primer in the pair.

The basics:

1. Avoid complementary clamps on the 3′ end of your primers, e.g. GC or GCGC, etc.
2. Measure and adjust your primer and template concentrations to recommended values.
3. Use touchdown PCR (temperature gradient) to take guesswork out of annealing temperatures.
4. Use high-fidelity DNA polymerases, e.g. Pfu.

To determine the amount of water to add to the lyophilized primer simply multiply the number of nmol of primer in the tube by 10. That will be the amount of water to add to make a 100 µM primer stock. For example, if there are 38.2 nmol of primer a 100 µM primer stock is created by adding 382 µl of water.

Avogadro’s number is a very important relationship to remember: 1 mole = 6.022×1023 6.022 × 10 23 atoms, molecules, protons, etc. To convert from moles to atoms, multiply the molar amount by Avogadro’s number. To convert from atoms to moles, divide the atom amount by Avogadro’s number (or multiply by its reciprocal).

First, identify the concentration required for your working stock. For primers, this typically ranges from 10–100 µM, and for probes, from 2–10 µM.

Add equal volume (eg. 10 ul) of of 1 mM DIG-dUTP. THEN add water to 10 vol (=100 ul; add 51 ul): final concentration each dNTP = 1 mM; final concn DIG-dUTP = 0.1 mM, and of dTTP = 0.9 mM.

1 to 100 ng cDNA

The molar concentration (molarity) is always written with a big “M” such as in micromolar – μM. This unit refers to the number of oligo molecules per unit of volume. For example, one might purchase 5 nanomoles (nmol) of probe delivered in solution at 100 micromolar (µM) concentration.

Molar concentration (also called molarity, amount concentration or substance concentration) is a measure of the concentration of a chemical species, in particular of a solute in a solution, in terms of amount of substance per unit volume of solution. …

Some medical tests report results in nanomoles (nmol). A mole is an amount of a substance that contains a large number (6 followed by 23 zeros) of molecules or atoms. A nanomole is one-billionth of a mole.

Some of the more common concentration units are:

• Mass per unit volume.
• Percent by mass.
• Percent by volume.
• Molarity.
• Normality.
• Molality.
• Parts per million (ppm).
• Parts per billion (ppb) This works like above, but we multiply by one billion (109: