PCR Mastermixes and Primer Design

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Questions and Answers

What is the primary purpose of the Polymerase Chain Reaction (PCR)?

To make many copies of a specific DNA sequence (correct)
To synthesize proteins from specific genes
To cut DNA into smaller fragments
To identify all genes in a genome

What components, other than the DNA template, are typically included in a PCR mastermix?

DNA polymerase, dNTPs, buffer, MgCl2, and primers

The formula for calculating the melting temperature (Tm) of a primer is Tm = 4(G + C) + 2(A + T)°С. In this formula 'G' and 'C' represent the number of ______ bases in the primer sequence.

guanine/cytosine

The annealing temperature is not critical for successful PCR amplification.

False (B) Signup and view all the answers

Why is it recommended to prepare a mastermix with slightly more reactions than needed for a PCR experiment?

To compensate for potential pipetting errors or loss of volume (C) Signup and view all the answers

Match the following PCR reagents with their function:

DNA Polymerase = Enzyme that synthesizes new DNA strands Primers = Short DNA sequences that initiate DNA synthesis dNTPs = Building blocks of DNA MgCl2 = Cofactor for DNA polymerase activity Signup and view all the answers

The size of human genome is aproximately 3.2 million base pairs

False (B) Signup and view all the answers

In PCR, what is the purpose of the initial denaturation step?

To separate the double stranded DNA into single strands Signup and view all the answers

The annealing temperature for primers is calculated to ensure the primers bind efficiently to the DNA template. If the temperature is too low, primers are not specific and can cause ______.

non-specific amplification Signup and view all the answers

What is the purpose of the heated lid on a thermal cycler?

To prevent condensation on the inside of the tube lids (A) Signup and view all the answers

Flashcards

What is PCR?

The polymerase chain reaction enables researchers to find specific gene sequences of interest and make copies of it.

PCR Primers

Primers are designed by the researcher, and their sequence must be specific to the gene sequence being targeted for amplification, typically about 20 nucleotides in length.

Steps to set up PCR

Primers are designed and synthesized. 2. Annealing temperature is calculated. 3. DNA is isolated and diluted. 4. PCR reagents are purchased. 5. Protocol is optimized. 6. A master mix is prepared. 7. Reactions are set up in a thermal cycler. 8. The cycler is programmed.

PCR Master Mix

A master mix is a pre-mixed solution containing all the necessary reagents for PCR, except for the DNA template. This simplifies setup, reduces pipetting errors, and improves reliability.

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Thermal Cycler

A thermal cycler is used in PCR to automatically cycle through different temperatures required for DNA denaturation, primer annealing, and DNA extension.

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Study Notes

Week 3 introduces PCR and the preparation of mastermixes.

Outcomes for Week 3

Designed to provide an understanding of PCR and the use of PCR mastermixes.

Background on PCR

The human genome size is approximately 3.2 billion base pairs.
Identifying a specific gene sequence is challenging due to the genome size.
PCR enables researchers to find and make copies of specific gene sequences of interest.
Primers are designed by the researcher and are about 20 nucleotides in length.
Proper primer length and sequence are crucial for successful amplification.
The melting temperature (Tm) formula is Tm = 4(G + C) + 2(A + T)°C.

Setting Up PCR

PCR setup requires primers to be designed and chemically synthesized.
Calculate the annealing temperature for the primers.
Isolate and dilute the DNA to create an appropriate working stock concentration.
PCR reagents are typically purchased as stocks, such as 10x buffer, 10mM dNTPs, 25 or 50mM MgCl2, and Taq polymerase at 5U/µl.
Some PCR buffers contain MgCl2, which means additional MgCl2 might not be needed.
Determining the appropriate final concentration of each reagent is achieved by optimizing the PCR protocol.
A mastermix made is able to amplify all samples.
Reactions are set up and placed in a thermal cycler.
Parameters are programmed into the thermal cycler and number of cycles.

PCR Mastermix

Mastermixes are used because PCR requires small volumes of reagents, which can lead to pipetting errors,.
Mastermixes are a mixture of the shared ingredients for PCR, which makes eases use.
All PCR reagents except the DNA template are combined in a single tube.
The mastermix should have more reactions than required to compensate for mistakes during pipetting.
A mastermix saves time and increases reliability.

The Biorad T100 Thermal Cycler

PCR samples are placed in the block.
The cycler is programmed.
The thermal cycler lid is heated to allow samples to be heated efficiently, preventing water condensation.

PCR Reaction Amplification

In the first cycle, DNA strands are separated and amplified into 2 DNA molecules.
In the second cycle, 2 DNA molecules amplify into 4 DNA molecules.
Subsequent cycles lead to exponential DNA amplification.
After 30 cycles, about 1 billion copies of the original sequence is made, or 2^30.