Lab Exam 2 - DNA Flashcards
polymerase chain (PCR)
Used to copy segments of DNA and make large amounts
3 stages of PCR
Denaturation, Annealing, Elongation/Extension
Denaturation process ( 1st step in PCR)
High temp (95c) is applied to a reaction to separate double stranded template DNA into 2 single strands
Annealing process
DNA primers (short complimentary pieces of DNA) are annealed to the start and end of an area to be replicated
What is template DNA
Starting DNA
Annealing function (2nd step in PCR)
Done at lower temp (50C) , to copy specific regions of DNA
Extension/Elongation ( 3rd step in PCR)
Final step in PCR , uses Tap polymerase and DNA nucleotides
Elongation process
Taq polymerase is activated , taq finds location of primers bound to starting template DNA , then synthesizes a complimentary DNA molecule by assembling DNA nucleotides
How is Taq polymerase activated
Exposed to high temp (72c)
How are DNA nucleotides used in PCR
Used by taq to synthesize complimentary DNA molecules with the assembly of DNA nucleotides
(Assembled with sequence info from template DNA)
SYBR GREEN
Under ultraviolet/blue light it fluoresces , making DNA bands visible
Applied to gel , binds to DNA
DNA extraction steps
- Mortar + pestle - breaks down membrane
- add saline (NaCl) - neutralizes DNA , bonds w negative electrons
- Cheesecloth - filter debris
- Add SDS solution - breaks down cell membrane
- Add Ethanol - precipitation, DNA becomes solid allowing for extraction
Saline (NaCl) use in Dna extraction
Bonds with negative electrons, neutralizing dna
SDS solution in Dna extraction
Disrupts plasma membrane ( after saline and cheesecloth)
Ethanol in Dna extraction
Causes precipitation , allowing dna to become solid and ready for extraction
Agragose
Sugar used to create a gel (extracted from seaweed)
TBE (step 2 in electrophoresis)
Conductive ph buffer , covers hardened gel in chamber
Dna loading buffer
Added to Dna so its easier to see/load
Made of sucrose and coloring
Net charge of DNA
Negative
DNA after electrical voltage is applied to gel
Dna migrates down towards positively charged electrode
SYBR GREEN
Causes Dna to fluoresce when placed under blue/ultraviolet light
(Binds to dna)
Gel Electrophoresis
Separates DNA fragments
Gel place in chamber with electrical voltage (negative electrodes near wells and positive on opposite side)
Dna migrates down towards positively charged electrode
