Alu PCR Lab http://emmynam.weebly.com/alu-pcr-lab.html
Hypothesis: am i going to have the allele (aloe) insert or not heterozygous or not
Vocabulary:
Heterozygous: Of, or pertaining to an individual (or a condition in a cell or an organism) containing two different alleles for a particular trait. Having dissimilar alleles that code for the same gene or trait.
Homozygous: Only have the same type of allele for a specific trait. Can mean having two Alu genes and also not having either. Homozygous as long as both traits are the same.
Allele: An allele is one of the possible forms of a gene. Most genes have two alleles, a dominant allele and a recessive allele. If an organism is heterozygous for that trait, or possessed one of each allele, then the dominant trait is expressed.
Genome: The complete set of genes in an organism. Also, the total genetic content in one set of chromosomes.
Genotype: Genotype is the information contained within two alleles. Genotype is the genetic makeup of an organism and it results in some of the physical characteristics of that organism. Genotypes can only be determined by biological tests, not observations.
Phenotype: The physical appearance of an organism as distinguished from its genetic makeup. The phenotype of an organism depends on which genes are dominant and on the interaction between genes and environment.
Heterozygous: Of, or pertaining to an individual (or a condition in a cell or an organism) containing two different alleles for a particular trait. Having dissimilar alleles that code for the same gene or trait.
Homozygous: Only have the same type of allele for a specific trait. Can mean having two Alu genes and also not having either. Homozygous as long as both traits are the same.
Allele: An allele is one of the possible forms of a gene. Most genes have two alleles, a dominant allele and a recessive allele. If an organism is heterozygous for that trait, or possessed one of each allele, then the dominant trait is expressed.
Genome: The complete set of genes in an organism. Also, the total genetic content in one set of chromosomes.
Genotype: Genotype is the information contained within two alleles. Genotype is the genetic makeup of an organism and it results in some of the physical characteristics of that organism. Genotypes can only be determined by biological tests, not observations.
Phenotype: The physical appearance of an organism as distinguished from its genetic makeup. The phenotype of an organism depends on which genes are dominant and on the interaction between genes and environment.
Materials:
1. 9% saline solution
2. Micro-pipettes, tips
3. Waste container
4. Micro-centrifuge
5. Micro-centrifuge tubes
6. PCR tubes
7. Agarose
8. 1xTAE
9.Gel box
10. Load Dye
12. Chelex
13. Primer Mix
14. Master Mix
15. Water
+ control DNA
16. Microwave
17. Electronic balance
18. Gel chambers and Molds
19. Epindorphs
1. 9% saline solution
2. Micro-pipettes, tips
3. Waste container
4. Micro-centrifuge
5. Micro-centrifuge tubes
6. PCR tubes
7. Agarose
8. 1xTAE
9.Gel box
10. Load Dye
12. Chelex
13. Primer Mix
14. Master Mix
15. Water
+ control DNA
16. Microwave
17. Electronic balance
18. Gel chambers and Molds
19. Epindorphs
Procedure:
1. Swirl 10mL of saline solution in mouth
2. spit the solution into a cup and swirl to mix cells
3. Move 1000 - 1500 uL of solution to a labeled micro-centrifuge tube
4. Spin tube in micro-centrifuge to retrieve cells from solution
5. Pour off the supernatant, but leave about 100uL to cover the cell pellet
6. Resuspend the cells
7. Add 50 uL of the suspension to a labeled Chelex tube
8. Place the tube into a termo-cycler for 10 minutes on the 99 Celsius temp.
9. Rack/ mix the tube's contents and then place in micro-centrifuge for 10 minutes
10. Transfer 50 uL of the supernatant from the tube in the previous step to a new tube labeled 'DNA'
Keep the PCR tube on ice
11. Obtain a PCR tube and add 20 uL of Master Mix
12. Add 20 uL of Primer Mi
13. Add 10 uL of extracted DNA to PCR tube
14. Place the tube into the Thermo-Cycler
15. Retrieve the tube and place it into the micro-centrifuge for 10 seconds
16. Add 5 uL of loading dye
17. Load 15 - 20 uL of the mixture to the Gel.
1. Swirl 10mL of saline solution in mouth
2. spit the solution into a cup and swirl to mix cells
3. Move 1000 - 1500 uL of solution to a labeled micro-centrifuge tube
4. Spin tube in micro-centrifuge to retrieve cells from solution
5. Pour off the supernatant, but leave about 100uL to cover the cell pellet
6. Resuspend the cells
7. Add 50 uL of the suspension to a labeled Chelex tube
8. Place the tube into a termo-cycler for 10 minutes on the 99 Celsius temp.
9. Rack/ mix the tube's contents and then place in micro-centrifuge for 10 minutes
10. Transfer 50 uL of the supernatant from the tube in the previous step to a new tube labeled 'DNA'
Keep the PCR tube on ice
11. Obtain a PCR tube and add 20 uL of Master Mix
12. Add 20 uL of Primer Mi
13. Add 10 uL of extracted DNA to PCR tube
14. Place the tube into the Thermo-Cycler
15. Retrieve the tube and place it into the micro-centrifuge for 10 seconds
16. Add 5 uL of loading dye
17. Load 15 - 20 uL of the mixture to the Gel.
Conclusion/Results: My genotype was +- (positive, negative)
My reflection:
It was very interesting to learn about micro pipetting and my own genotypes. The lab itself was challenging, because there was a misunderstanding in how we were supposed to follow the instructions, and that caused a delay. But all and all it was a satisfying experience. I'm glad I now know that I have an Alu type of negative negative.
It was very interesting to learn about micro pipetting and my own genotypes. The lab itself was challenging, because there was a misunderstanding in how we were supposed to follow the instructions, and that caused a delay. But all and all it was a satisfying experience. I'm glad I now know that I have an Alu type of negative negative.