Can you use the same primers for PCR and RT PCR?

Can you use the same primers for PCR and RT PCR?

There is no difference. For a reliable quantification by real-time PCR it is very important that the primers really amplify exclusively the specific product and that this amplification runs with almost optimum efficiency.

How do you know if primer is contaminated?

The best way to check whether your primers are contaminated would be to set up a PCR with the same primers but using fresh aliquots of all the other PCR component reagents(ie fresh aliquots of PCR grade H2O , Taq polymerase, dNTPs, Mg2+ etc.).

What is a primer assay?

QuantiTect Primer Assays are for gene expression analysis using one-step and two-step qRT-PCR with SYBR Green detection. They are highly suited for applications such as: Validation of siRNA-mediated gene knockdown.

How do you dilute primers for RT PCR?

This can usually be found on the tube itself or the primer sheet supplied with the order. For every 1 nmoles, add 10 μL of PCR-grade water. For example, if a primer states 19.4 nmoles, then add 194 μL of PCR-grade water. Mix the solution by vortexing to reconstitute the primers.

What type of primers are used in RT-PCR?

There are three RT primer options – oligo-dT primers (typically 13–18mers), random oligomers (such as hexamers, octamers, or nonamers), and gene-specific primers. One-step RT-PCR is always performed with gene-specific primers as the downstream PCR primer is also the primer for reverse transcription.

Is RT-PCR same as PCR?

RT–PCR is a variation of PCR, or polymerase chain reaction. The two techniques use the same process except that RT–PCR has an added step of reverse transcription of RNA to DNA, or RT, to allow for amplification.

How can PCR contamination be prevented?

Separating pre- and post-amplification areas is key to preventing contamination. Prepare your PCR master mix in a template-free room (see next bullet), using reagents that never come into contact with potential sources of contamination. Maintain a separate area for analyzing PCR amplicons.

What is a PCR primer?

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. Primers are also referred to as oligonucleotides.

What is primer used for?

A primer (/ˈpraɪmər/) or undercoat is a preparatory coating put on materials before painting. Priming ensures better adhesion of paint to the surface, increases paint durability, and provides additional protection for the material being painted.

How do you make a stock primer solution?

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.

Can I dilute primer with water?

Never have problem occur before for all my primers. i have used RNase free water for primer dilution. you can use distilled water for primer dilution. Water dissolves the oligos and you can store them in -20 for long.