Chelex Lab Report
Chelex Extraction and PCR Amplification of the Amelogenin Gene For The Purpose of Gender Determination.
Abstract
The amelogenin gene is a protein involved in the development of the enamel matrix. It is located on both the X and Y chromosomes in humans and can be used as a way to distinguish between males and females. Gender determination of a human has important applications in forensic science, prenatal diagnosis, etc. This can be done by Polymerase Chain Reaction (PCR) followed by gel electrophoresis. The presence of two bands of DNA indicates that the sample is from a male while the presence of one band of DNA indicates that the sample is from a female (sasaki and shimokawa, 1995). In this experiment, Chelex was used to extract nucleic …show more content…
The cheek cells were re-suspend in the Chelex, after which 500µl of the re-suspended solution was transferred into a 1.5ml eppendorf tube. The tube was incubated in boiling water for 10 minutes, centrifuged again to pellet the Chelex beads and 100µl of the supernatant was transferred to a fresh eppendorf tube. In order to access the purity and concentration of the DNA sample, the NanoDrop 2000 was used with the accompanying Nanodrop 2000/2000c operating software. Reagents Amount(µl)
Diluted template DNA stock (DNA diluted using TEA buffer to obtain a concentration of 10ng/µl) 2.50
Taq 2x Master Mix 12.50
Amelogenin Forward Primer(10µM) [primer sequence: GCTCTGATGGTTGGCC] …show more content…
To obtain a 1x concentration both SYBR-Green I and the loading buffer, 3.1µl of both were added to the PCR samples (25µl). 20µl of this mixture was loaded onto the Agarose gel. The gel was 1.5% agarose in 1x Tris-Acetate-EDTA (TAE; 40mM Tris-Acetate, 1mM EDTA, pH 8.3) and was run at 130 V for 60 minutes in a 1x TAE buffer. A 100bp ladder was used as a reference to compare the results to. The ladder was used because it reads from 100bp up to 1200bp fragments and we were expecting the amelogenin fragments to be around 542bp and 358bp for X and Y chromosomes respectively. Afterward, the gel image was captured on a Kodak Gel 100 image acquisition system which is equipped with green filter for SYBR Green
Abstract
The amelogenin gene is a protein involved in the development of the enamel matrix. It is located on both the X and Y chromosomes in humans and can be used as a way to distinguish between males and females. Gender determination of a human has important applications in forensic science, prenatal diagnosis, etc. This can be done by Polymerase Chain Reaction (PCR) followed by gel electrophoresis. The presence of two bands of DNA indicates that the sample is from a male while the presence of one band of DNA indicates that the sample is from a female (sasaki and shimokawa, 1995). In this experiment, Chelex was used to extract nucleic …show more content…
The cheek cells were re-suspend in the Chelex, after which 500µl of the re-suspended solution was transferred into a 1.5ml eppendorf tube. The tube was incubated in boiling water for 10 minutes, centrifuged again to pellet the Chelex beads and 100µl of the supernatant was transferred to a fresh eppendorf tube. In order to access the purity and concentration of the DNA sample, the NanoDrop 2000 was used with the accompanying Nanodrop 2000/2000c operating software. Reagents Amount(µl)
Diluted template DNA stock (DNA diluted using TEA buffer to obtain a concentration of 10ng/µl) 2.50
Taq 2x Master Mix 12.50
Amelogenin Forward Primer(10µM) [primer sequence: GCTCTGATGGTTGGCC] …show more content…
To obtain a 1x concentration both SYBR-Green I and the loading buffer, 3.1µl of both were added to the PCR samples (25µl). 20µl of this mixture was loaded onto the Agarose gel. The gel was 1.5% agarose in 1x Tris-Acetate-EDTA (TAE; 40mM Tris-Acetate, 1mM EDTA, pH 8.3) and was run at 130 V for 60 minutes in a 1x TAE buffer. A 100bp ladder was used as a reference to compare the results to. The ladder was used because it reads from 100bp up to 1200bp fragments and we were expecting the amelogenin fragments to be around 542bp and 358bp for X and Y chromosomes respectively. Afterward, the gel image was captured on a Kodak Gel 100 image acquisition system which is equipped with green filter for SYBR Green