PCR Lab: Polymerase Chain Reaction Lab

Objective: To collect a DNA sample from the cells inside a persons mouth and use this sample to explore Polymerase Chain Reaction (PCR) Harvest cheek cells Transfer the cells to a microfuge tube
The solution inin the The solution the microfuge contains microfuge contains Chelex Mg++ Chelex Mg++

"ource ofof DN "ource DN

Helps reduce Helps reduce the degradation the degradation ofof DNA DNA

Centrifuge

#O$L %% #O$L minutes minutes

#oiling l&ses #oiling l&ses the cell the cell membrane membrane an' releases an' releases DN DN( (

"e!arates DN cell "e!arates DN an' an' cell organelles b& 'ensit& ( ( organelles b& 'ensit&

DNA isis inin the supernate DNA the supernate li!uid top li!uidon on top

Grab DN

sam!le Remo"e the DNA from the li!uid atat the top Remo"e the DNA from the li!uid the top ofof the microfu#e tube toto PCR the microfu#e tubeTransfer Transfer PCR tube . . tube PCR tube isis ready for the Thermocycler PCR tube ready for the Thermocycler

PCR Thermoc&cler: )olecules in each PCR tube:

$our DNA strands (to be copied)

%ree DNA nucleotides

Ta! Polymerase (bacterial heat stable DNA polymerase)

Primers (frames the locus&site to be copied)

'uffer (stabili(es p) at * + ,hich is the optimum for en(ymes in this lab)

PCR "te!s in Thermoc&cler

(repeated for -+ hours).

/ Heating. Denatures and Separates DNA strands so the Taq Polymerase can attach and make copies *( nnealing: Primers anneal attach to DNA to show Taq Polymerase (DNA Polymerase) the starting points. 0 +longation: Taq Polymerase attaches at Primers and mo es along the DNA strands to make more copies o! the original genes "y attaching !ree DNA nucleotides according to the #ase Pair $ule% A & T ' & (.

After the PCR thermocycler has amplified (made copies) your DNA1 students load DNA sample into the #el ,ells 2ell 3/ ,ill be used for the mar4er 5tudents ,ill use the remainin# ,ells Record student 6D for each ,ell number

Gel with DNA

Gel after run the test

)D*A+ 'el% ,A$-*$ "ase pairs on le!t in lane . /. Three Su"0ects Data in red%
__ 1000 00 !00 300 200 100 Left-over primers Ignore this line Alu- / AluHomo Alu+ / Alu+ Homo+ Alu- / Alu+ Heterozygous