Purifying DNA to Estimate its Purity using PCR Amplification of VNTR to Load and Run Agarose Gel
Introduction
The study of this experiment was the Dopamine transporter gene. This gene is associated with different brain disorders like bipolar, as well as certain behavioural traits such as ADHD.[1] Dopamine transporter gene is a presynaptic plasma protein containing different VNTRs in it’s UTR and plays an important role in restricting the activity of dopamine by rapid reuptake into the presynaptic neuron. DAT is part of Na+ and Cl- dependent family with the addition of other neurotransmitter transporters such as, GABA, serotonin, glycine, and norepinephrine transporters. The 3' UTR of this gene contains a 40 bp tandem repeat.[4] Dopamine is required for central nervous system functions normality, these functions include, motor activity, cognition and affect.[2] Drugs can disrupt the activity of the dopamine transporter gene such as amphetamine and cocaine and are linked due to the VNTR in the DAT gene. The DAT gene contains fifteen coding exons which are located in chromosome 5 and encodes DAT protein. DAT transports dopamine across the cell membrane in a movement known as coupling towards the sodium ions that move into the cell from a high concentration to a low concentration.[3] The aim of the practical was to purify genomic DNA from buccal cells using DNA quantitation, PCR amplification of VNTR and agarose gel. Materials and Methods 10ml of 0.9% saline was swished in the mouth for about 20 seconds then collected in a sterile 50ml Falcon tube which was then centrifuged at 2000 rpm for 10 minutes. With the use of a pipette, the liquid was put into bleach pots to be discarded while the pellet at the bottom was saved. To get the resuspension of cells the base was flicked and 500ul of resuspended chelex beads were added to break down the DNA. After pipetting up and down a few times to ensure no clumps were left, 500ul of the solution was placed into a 1.5ml screw cap eppendorf and boiled for 10 minutes in a hot block followed by ice bath for a further 5 minutes. The tube span 13,000 rpm for 3 minutes in a microfuge. The supernatant was then transferred into a clean eppendorf tube and placed in an ice bucket and stored at -20∘c. 1:10 dilution of DNA sample was made by adding the DNA sample to a 1.5ml eppendorf tube as well as 1.35ml of water. 1.5ml of the diluted sample was transferred to a UV cuvette. The spectrophotometer was turned to zero and absorbance readings were taken, 280 nm for protein/phenol and 260 nm for the DNA sample. Calculations were complete using the following equation: DNA concentration (ug/ml) = OD260 x 50 x L x RDF DNA purity = OD260 / OD280 A clean sample would have a ratio between 1.8 and 2.0. A mastermix was prepared for 5 PCR reactions with the reaction volume 50ul. Reagents were added to the tube …show more content…
As well as DNA, RNA molecules are also able to absorb UV light at 260nm as they contain amino acids, therefore, they both contributed to the total result.
Agarose Gel Electrophoresis of DAT VNTR PCR Products
DNA denatures causing a smear as when the gene is stuck to an enzyme, the DNA fragments can be seen in a smear on an agarose gel after performing electrophoresis.[5] For this test, a small sample of DNA that had been isolated was loaded into a well in the agarose gel which was then exposed to an electric field. DNA is negatively charged so moved towards the anode. Due to small fragments of DNA moving faster, the DNA was separated in size. This can be seen in figure 1.
IMG_0698.JPG
Figure 1
Image of agarose gel taken by a UV transilluminator.
A few PCR bands can be seen in figure 1. …show more content…
However, some affecting factors were unavoidable and can be classed as human error. In future, these small mistakes can be avoided by just simply working at a steady pace and ensuring all equipment is working correctly such as the timer on the powerpack. Furthermore, practice should be done for pipetting samples into the agarose gel so that there is no hesitation, overfilling or underfilling during the procedure. If these slight errors were not to contribute to the experiment next time, I am certain the results will portray clear PCR bands for a more detailed
Introduction
The study of this experiment was the Dopamine transporter gene. This gene is associated with different brain disorders like bipolar, as well as certain behavioural traits such as ADHD.[1] Dopamine transporter gene is a presynaptic plasma protein containing different VNTRs in it’s UTR and plays an important role in restricting the activity of dopamine by rapid reuptake into the presynaptic neuron. DAT is part of Na+ and Cl- dependent family with the addition of other neurotransmitter transporters such as, GABA, serotonin, glycine, and norepinephrine transporters. The 3' UTR of this gene contains a 40 bp tandem repeat.[4] Dopamine is required for central nervous system functions normality, these functions include, motor activity, cognition and affect.[2] Drugs can disrupt the activity of the dopamine transporter gene such as amphetamine and cocaine and are linked due to the VNTR in the DAT gene. The DAT gene contains fifteen coding exons which are located in chromosome 5 and encodes DAT protein. DAT transports dopamine across the cell membrane in a movement known as coupling towards the sodium ions that move into the cell from a high concentration to a low concentration.[3] The aim of the practical was to purify genomic DNA from buccal cells using DNA quantitation, PCR amplification of VNTR and agarose gel. Materials and Methods 10ml of 0.9% saline was swished in the mouth for about 20 seconds then collected in a sterile 50ml Falcon tube which was then centrifuged at 2000 rpm for 10 minutes. With the use of a pipette, the liquid was put into bleach pots to be discarded while the pellet at the bottom was saved. To get the resuspension of cells the base was flicked and 500ul of resuspended chelex beads were added to break down the DNA. After pipetting up and down a few times to ensure no clumps were left, 500ul of the solution was placed into a 1.5ml screw cap eppendorf and boiled for 10 minutes in a hot block followed by ice bath for a further 5 minutes. The tube span 13,000 rpm for 3 minutes in a microfuge. The supernatant was then transferred into a clean eppendorf tube and placed in an ice bucket and stored at -20∘c. 1:10 dilution of DNA sample was made by adding the DNA sample to a 1.5ml eppendorf tube as well as 1.35ml of water. 1.5ml of the diluted sample was transferred to a UV cuvette. The spectrophotometer was turned to zero and absorbance readings were taken, 280 nm for protein/phenol and 260 nm for the DNA sample. Calculations were complete using the following equation: DNA concentration (ug/ml) = OD260 x 50 x L x RDF DNA purity = OD260 / OD280 A clean sample would have a ratio between 1.8 and 2.0. A mastermix was prepared for 5 PCR reactions with the reaction volume 50ul. Reagents were added to the tube …show more content…
As well as DNA, RNA molecules are also able to absorb UV light at 260nm as they contain amino acids, therefore, they both contributed to the total result.
Agarose Gel Electrophoresis of DAT VNTR PCR Products
DNA denatures causing a smear as when the gene is stuck to an enzyme, the DNA fragments can be seen in a smear on an agarose gel after performing electrophoresis.[5] For this test, a small sample of DNA that had been isolated was loaded into a well in the agarose gel which was then exposed to an electric field. DNA is negatively charged so moved towards the anode. Due to small fragments of DNA moving faster, the DNA was separated in size. This can be seen in figure 1.
IMG_0698.JPG
Figure 1
Image of agarose gel taken by a UV transilluminator.
A few PCR bands can be seen in figure 1. …show more content…
However, some affecting factors were unavoidable and can be classed as human error. In future, these small mistakes can be avoided by just simply working at a steady pace and ensuring all equipment is working correctly such as the timer on the powerpack. Furthermore, practice should be done for pipetting samples into the agarose gel so that there is no hesitation, overfilling or underfilling during the procedure. If these slight errors were not to contribute to the experiment next time, I am certain the results will portray clear PCR bands for a more detailed