The amount of each component for a PCR reaction is critical. The final concentration of each primer should be 0.2 uM, and the final concentration of dNTPs is 0.2 mM. If the final concentrations of these components are significantly off, your PCR reactions will not work. The stock concentration of primers is usually 10 uM and the stock concentration of dNTPs is usually 10 mM. PCR buffer is supplied as 10X and should be 1X in the final reaction volume. The amount of Taq DNA polymerase necessary will depend on the manufacturer. For our purposes, Taq polymerase is supplied at a concentration of 20 Units/ ul. Each PCR reaction should include 5 units of Taq polymerase. 1) Calculate the necessary volume to set up a single PCR reaction given the following: Assume you will use 2 ul of template DNA (e.g. isolated from a patient). You have the following primer stocks GAPDH Forward Primer, 10 uM stock GAPDH Reverse Primer, 10 uM stock SARS-CoV-2 Forward Primer, 10 uM stock SARS-CoV-2 Reverse Primer, 10 uM stock 10X PCR Reaction Buffer dNTPs, 10 mM stock Taq polymerase, 20 Units/uL stock The final reaction volume should be 30 ul 2) Now, calculate what is needed to run samples for five patients plus a negative, no-template control sample. In your notebook, plan the pooled reaction that would be required to run these six PCR reactions. Remember that a pooled reaction should only include components common to all reactions and that you need to mix enough for one extra reaction to account for errors in pipetting.
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
The amount of each component for a PCR reaction is critical. The final concentration of each primer should be 0.2 uM, and the final concentration of dNTPs is 0.2 mM. If the final concentrations of these components are significantly off, your PCR reactions will not work. The stock concentration of primers is usually 10 uM and the stock concentration of dNTPs is usually 10 mM. PCR buffer is supplied as 10X and should be 1X in the final reaction volume. The amount of Taq DNA polymerase necessary will depend on the manufacturer. For our purposes, Taq polymerase is supplied at a concentration of 20 Units/ ul. Each PCR reaction should include 5 units of Taq polymerase.
1) Calculate the necessary volume to set up a single PCR reaction given the following:
Assume you will use 2 ul of template DNA (e.g. isolated from a patient).
You have the following primer stocks
GAPDH Forward Primer, 10 uM stock
GAPDH Reverse Primer, 10 uM stock
SARS-CoV-2 Forward Primer, 10 uM stock
SARS-CoV-2 Reverse Primer, 10 uM stock
10X PCR Reaction Buffer dNTPs, 10 mM stock
Taq polymerase, 20 Units/uL stock
The final reaction volume should be 30 ul
2) Now, calculate what is needed to run samples for five patients plus a negative, no-template control sample. In your notebook, plan the pooled reaction that would be required to run these six PCR reactions. Remember that a pooled reaction should only include components common to all reactions and that you need to mix enough for one extra reaction to account for errors in pipetting.
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