BME100 s2017:Group4 W8AM L4
BME 100 Spring 2017 | Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help | ||||||||||||||||||||||||||||||||
OUR TEAMLAB 4 WRITE-UPProtocolMaterials
have the same forward primer and reverse primer
do, the samples will become cross-contaminated
PCR Reaction Sample List
HEATED LID: 100°C INITIAL STEP: 95°C NUMBER OF CYCLES: 25 (Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds and Extend for at 72°C for 30 seconds) FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C
Research and DevelopmentPCR - The Underlying Technology
The Template DNA is used as a guide to make the replicate DNA. Primers attach to sites on the DNA strands that are at either end of the segment that is being copied. The Taq DNA Polymerase reads the DNA code and attaches matching nucleotides to create DNA copies. The deoxyribonucleotides (dNTP’s) are genetic building blocks that are used to create the replicate DNA strands.
During the initial step at 95°C for 3 minutes, the DNA double helix separates, creating two single-stranded DNA molecules. During the denature step at 95°C for 30 seconds, the DNA double helix separates, creating two single-stranded DNA molecules. During annealing at 57°C for 30 seconds, the primer sequences bind to specific areas on the single-stranded DNA. During it's extension at 72°C for 30 seconds, the DNA polymerase finds the primer and attaches to the DNA strand and begins to add complementary nucleotides onto the strand. During the final step at 72°C for 3 minutes, the DNA polymerase finishes adding to the DNA strand and falls off. During the final hold at 4°C, the DNA stand cools down to ensure that everything has recombined fully.
Adenine pairs with Thymine and Cytosine pairs with Guanine.
Base-pairing occurs during annealing and extension. In annealing, the base pairs of the primers bind with their complementary base pairs on the DNA strands. In extension, DNA polymerase attaches complementary base pairs to the base pairs on the DNA strand.
SNP Information & Primer DesignBackground: About the Disease SNP
The numerical position of the SNP is 117587799. The non-disease forward primer was found to be 5’- A G A A G G T G G A A T C A C A C T G A, while the non-disease reverse primer was found to be 5’- C A T T A T T T A T A G T T C T T A A A. The disease forward primer was found to be 5’- A G A A G G T G G A A T C A C A C C G T and the disease reverse primer was 5’- C A T T A T T T A T A G T T C T T A A T. When ran through the In-Silico PCR website, the non-disease primer resulted in 220bp sequence from the chromosome indicating the primer worked because it is a known sequence. The disease- specific primer designed resulted in no matches, because of a mutation in the DNA sequence, which matches what was expected of the diseased primer.
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