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Polymerase Chain Reaction (PCR) Amplification
Gel Electrophoresis of Amplified PCR Samples
This experiment focuses on utilizing polymerase chain reaction (PCR) to amplify a specific DNA chain, specifically the PV92 region of chromosome 16, aiming to determine the presence of the Alu sequence within that region.
PCR will be employed to amplify the target DNA sequence extracted from hair follicles.
The experiment involves three main steps of PCR:
Results will be analyzed by performing gel electrophoresis on the amplified DNA sequence. The outcomes will fall into three categories based on the DNA size of the PCR product: Genotype | DNA Size of PCR Product (bp) Homozygous (+/+) | 941 Homozygous (-/-) | 641 Heterozygous (+/-) | 941-641 Table 2.1 - Classification of Genotype versus the DNA size of PCR product
Polymerase Chain Reaction (PCR) is a method employed to amplify specific segments along a DNA strand, with the capability to amplify up to 10 kilo base pairs.
This process involves thermo cycling, comprising three essential steps: denaturation, primer annealing, and extension of new DNA strands.
During denaturation, the mixture is subjected to high temperatures, leading to the separation of the parent strand DNA.
Subsequently, in the annealing process, the mixture is cooled, and primers actively seek complementary sequences in the DNA, forming hydrogen bonds. The extension process follows, involving a reheating to 72°C, the optimal temperature for Taq polymerase to synthesize new complementary DNA strands, initiating from the 3’-OH ends of the primers. These three steps are iterated through 40 cycles, as detailed in Section 7, Question 4.
3.2 Gel Electrophoresis of Amplified PCR Samples
Figure 3.1 – Gel Electrophoresis
In this experiment, Gel Electrophoresis was employed to identify target sequences. Gel Electrophoresis operates based on the principles of size and charge, facilitating the separation of fragments. Upon loading samples into the gel wells, an electric current is applied through electrodes at both ends. As DNA fragments carry a negative charge, they migrate across the gel, being attracted to the positive end.
The rate of movement is determined by fragment size, with larger fragments encountering more resistance and moving more slowly through the gel matrix. This allows us to discern the size of DNA fragments and identify the presence of the Alu insertion.
PCR Amplification:
Gel Electrophoresis:
Procedures:
For this part of the experiment, collect 2 strands of hair for each group to prepare the DNA template for the subsequent lab session. Utilize the hair bulb and InstaGene matrix plus protease for DNA template extraction from the hair follicle.
Step 2a: Obtain a PCR tube containing 10 μL of yellow PCR master mix. Step 2b: Use a micropipette to transfer 10 μL of the DNA template from the supernatant in the screwcap tube into the PCR tube containing the master mix. Step 2c: Ensure thorough mixing by pipetting up and down 2 to 3 times, avoiding the formation of air bubbles, especially at the bottom of the PCR tube. Step 2d: Place the PCR tube in the MyCycler thermal cycler. Step 2e: Allow 40 cycles of amplification to take place within the 3-hour timeframe.
Laboratory Calculations and Formulas:
No specific calculations or formulas are explicitly mentioned in the provided information. However, it's essential to be mindful of the volumes being used in pipetting, especially when transferring the DNA template into the PCR tube. Precise and accurate pipetting is crucial for obtaining reliable experimental results.
Table 5.1 - Materials for PCR Amplification
Material | Quantity/Volumes |
---|---|
InstaGeneTM matrix plus protease | 200 μL |
Complete master mix (with primers) | 1 x tube (on ice) |
Strands of hair (from the same person) | 2 x strands |
Foam micro test tube holder | 1 |
Vortex mixer | 1 |
Stopwatch timer | 1 |
PCR tube | 1 |
Capless micro test tube | 1 |
P-20 Micropipet | 1 |
Pipet Tips | As needed |
Screwcap tube | 1 |
Water bath at 56 °C | 1 |
Water bath at 100°C | 1 |
Centrifuge | 1 |
MyCycler Thermal Cycler | 1 |
Table 5.2 - Materials for Gel Electrophoresis
Material | Quantity/Volumes |
---|---|
Student's PCR sample | As needed |
PV92 XC DNA loading dye | 10 μL |
TAE electrophoresis buffer | 275 mL |
MMR (DNA standard) | 10 μL |
PV92 homozygous (+/+) control sample | 10 μL |
PV92 homozygous (−/−) control sample | 10 μL |
PV92 heterozygous (+/−) control sample | 10 μL |
Fast BlastTM DNA stain (100x) | 120 mL |
Warm tap water | 1.5 - 2 L |
The provided laboratory setup and procedures involve essential steps for PCR amplification and gel electrophoresis, utilizing specified materials and equipment. Precise pipetting and adherence to the outlined procedures are crucial for successful experimentation.
Steps | Description |
Denaturation | Heating DNA template to approximately 94°C, separating double-stranded DNA by breaking bonds. Taq DNA polymerase withstands this high temperature. |
Annealing | Conducted at 60°C, primers attach to complementary sequences on each strand of the DNA template. Optimal temperature for primer binding. |
Extension | Temperature raised from 60°C to 72°C for optimal polymerase activity. Taq DNA polymerase adds nucleotides to create a complementary copy of the DNA template. |
Exploring PCR Amplification and Gel Electrophoresis: A Comprehensive Laboratory Study. (2024, Feb 28). Retrieved from https://studymoose.com/document/exploring-pcr-amplification-and-gel-electrophoresis-a-comprehensive-laboratory-study
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