Bio208 Genetics Lab
BIO208 Genetics Lab
What is PCR and How Does It Work?
BIO208 Genetics Lab: What is PCR and How Does It Work?
EXPERIMENT OBJECTIVE: The objective of this experiment is for students to gain handson experience in the principles and practice of Polymerase Chain Reaction (PCR).
BACKGROUND:
Polymerase Chain Reaction (PCR), discovered by Kary Mullis, has had an extraordinary impact on various aspects of biotechnology. PCR has revolutionized research and diagnostics-based molecular biology. PCR is a simple, accurate, and highly reproducible procedure. The technology introduced an important advantage to molecular biology. It provides the ability to start with a small amount of DNA and to be able to amplify it so that there will be a sufficient …show more content…
PCR is also used in genome projects for
DNA mapping and sequencing and is being applied to forensics, paternity determinations, as well as the determination of evolutionary relationships. In all these cases the DNA samples that are extracted are limited and PCR amplifies segments of DNA that become the subject for further analysis and study.
In a PCR reaction, the first step is the preparation of the DNA sample that is extracted from tissues or various biological sources. In PCR experiments, the DNA or gene to be amplified is referred to as the target and the synthetic oligonucleotides used are referred to as primers. A set of two primers (a forward and reverse primer) usually ranging between 20 and 45 nucleotides are chemically synthesized to correspond to the two ends of the gene to be amplified. Each primer binds to one of the two DNA strands and is the initiation point of the amplification. The primer concentrations are always in excess of the target gene to make possible subsequent priming. The exact nucleotide primer sequences for a …show more content…
(Background adapted from Edvotek protocols)
2
Figure 1: An overview of the PCR reaction.
BIO208 Genetics Lab
What is PCR and How Does It Work?
MATERIALS:
i) DNA template: (conc= 12.5ng/ul) ii) Primer 1 (12.5 μM) iii) Primer 2: (12.5 μM) iv) Primer 3: (12.5 μM)
v) Water vi) BIOMIX – contains Taq DNA polymerase, dNTPs, Mg2+, and buffering salts
Note: Primer pair 1,2 yields a product of ~ 550bp product while Primer pair 1,3 gives ~
1025bp product.
BIOMIX is a complete ready-to-use 2x reaction mix containing an ultrastable Taq DNA polymerase. Developed to perform PCR assays of many common genomic and cDNA templates, the user has simply to add water, template and primers. BioMix dramatically reduces the time required to set up reactions, thereby minimizing the risk of contamination.
Greater reproducibility is ensured, by reducing the number of pipetting steps that can lead to errors. PROCEDURE:
DAY 1:
1) Label tubes 1-4. Make sure to use 0.2 ml thin walled PCR tubes for this step.
2) Add reagents as shown in the table below.
Page
3
3) Add one drop of mineral oil to each tube to avoid sample from condensing on the top of
What is PCR and How Does It Work?
BIO208 Genetics Lab: What is PCR and How Does It Work?
EXPERIMENT OBJECTIVE: The objective of this experiment is for students to gain handson experience in the principles and practice of Polymerase Chain Reaction (PCR).
BACKGROUND:
Polymerase Chain Reaction (PCR), discovered by Kary Mullis, has had an extraordinary impact on various aspects of biotechnology. PCR has revolutionized research and diagnostics-based molecular biology. PCR is a simple, accurate, and highly reproducible procedure. The technology introduced an important advantage to molecular biology. It provides the ability to start with a small amount of DNA and to be able to amplify it so that there will be a sufficient …show more content…
PCR is also used in genome projects for
DNA mapping and sequencing and is being applied to forensics, paternity determinations, as well as the determination of evolutionary relationships. In all these cases the DNA samples that are extracted are limited and PCR amplifies segments of DNA that become the subject for further analysis and study.
In a PCR reaction, the first step is the preparation of the DNA sample that is extracted from tissues or various biological sources. In PCR experiments, the DNA or gene to be amplified is referred to as the target and the synthetic oligonucleotides used are referred to as primers. A set of two primers (a forward and reverse primer) usually ranging between 20 and 45 nucleotides are chemically synthesized to correspond to the two ends of the gene to be amplified. Each primer binds to one of the two DNA strands and is the initiation point of the amplification. The primer concentrations are always in excess of the target gene to make possible subsequent priming. The exact nucleotide primer sequences for a …show more content…
(Background adapted from Edvotek protocols)
2
Figure 1: An overview of the PCR reaction.
BIO208 Genetics Lab
What is PCR and How Does It Work?
MATERIALS:
i) DNA template: (conc= 12.5ng/ul) ii) Primer 1 (12.5 μM) iii) Primer 2: (12.5 μM) iv) Primer 3: (12.5 μM)
v) Water vi) BIOMIX – contains Taq DNA polymerase, dNTPs, Mg2+, and buffering salts
Note: Primer pair 1,2 yields a product of ~ 550bp product while Primer pair 1,3 gives ~
1025bp product.
BIOMIX is a complete ready-to-use 2x reaction mix containing an ultrastable Taq DNA polymerase. Developed to perform PCR assays of many common genomic and cDNA templates, the user has simply to add water, template and primers. BioMix dramatically reduces the time required to set up reactions, thereby minimizing the risk of contamination.
Greater reproducibility is ensured, by reducing the number of pipetting steps that can lead to errors. PROCEDURE:
DAY 1:
1) Label tubes 1-4. Make sure to use 0.2 ml thin walled PCR tubes for this step.
2) Add reagents as shown in the table below.
Page
3
3) Add one drop of mineral oil to each tube to avoid sample from condensing on the top of