Lab: Genetics 1 (PCR and DNA Isolation)

central dogmas

DNA → RNA → proteins

purines

adenine and guanine, double rings, Pure As Gold

pyrimidines

cytosine and thymine (and uracil for RNA), one ring, CUT the Pyramid

DNA has

directionality 

ploidy 

expected number of sets of chromosomes per cell 

diploid

2n, somatic cells, have two copies of each chromosome

haploid

1n, gametes, have 1 copy of each chromosome

start codon

AUG

stop codon

UAA, UAG, UGA

lysis buffer

breaks open cell

potassium acetate

stabilizes DNA

isopropanol

precipitates DNA

ammonium acetate

washes DNA

glycoblue

dyes pellet to make it visible

nuclease-free water

resuspends DNA

How is the concentration of DNA measured?

with a microvolume (nanodrop) spectrophotometer, DNA absorbance is measured at a lambda max value of 260 nm

What are the reagents used in PCR

• Taq polymerase

• Foward and reverse primers

• nucleotides (dNTPs)

• magnesium chloride 

Taq polymerase

DNA-polymerase that works at high temps

forward and reverse primers 

• synthetic DNA fragments (20-30 bps) 

• complimentary to certain gene or region 

magnesium chloride

cations for Taq polymerasen

In PCR, what happens at 90 degrees C?

denaturation, DNA strands break apart

In PCR, what happens at 60 degrees C?

annealing, connecting primers to each strand

In PCR, what happens at 72 degrees C?

extension, Taq polymerase extends new nucleotides from primers

calculation for DNA amplificaton PCR

X x 2ⁿ

• X = starting number of DNA templates