Genetics Lab: PCR identification of Genotype (Kim)

what is the extraction buffer made of that we used for this experiment?

- 100 mM Tris, pH = 8

- 25 mM EDTA

- 250 mM NaCl

- 1% PVP-40

tissue homogenization

grind plant tissue in 300 microliters extraction buffer

cell lysis

after homogenization, add 350 more microliters of the extraction buffer and invert tube 3-6 times

what do you do after cell lysis step?

add 90 microliters of the 10% SDS

what does the 10% SDS do?

it denatures the proteins

to precipitate the SDS complexed protein, what mixture do you need to add?

250 microliters of KOAc (potassium acetate)

what temperate helps SDS function?

65 degrees C

what makes up the white precipitate after adding the potassium acetate?

denatured proteins/cell debris and other insoluble matter that is separated by the centrifuge

how much of the supernatent is transferred to a fresh tube after the potassium acetate is added? what else must be added to the tube?

650 microliters and 650 microliters of isopropanol

what is added to the tube to purify the sample?

500 microliters of 70% ethanol

for resuspension what is added?

50-100 microliters of water

dihydroflavanol 4-reductase (DFR)

essential enzyme in the anthocyanin biosynthesis pathway

what are the loss of function mutations in the DFR gene responsible for?

the anthocyaninless phenotype (anl) in many plant species

all nonpurple strains have an insertion of 384 bases in the fourth exon of DFR, this alters the reading frame and introduces a premature what?

stop codon

what are the mutations that occur in the DFR gene?

a frameshift mutation and a premature stop codon

Primer set CP is made of =

CF + PR

Primer set CN is made of =

CF + NR

all primer sequences go in what direction?

5' to 3'

how much of the primers did we add?

2 microliters

how much of the gDNA did we add?

8 microliters

how much of the PCR mix did we add?

10 microliters

what PCR mix did we use?

GoTaq 2x master mix

what is the initial temperature for denaturation during the PCR program test? and how long does it last?

94 degrees C, 120 seconds

what is the temperature for the normal denaturation step and how long does it last?

94 degrees C, 45 seconds

what is the temperature for the annealing step and how long does this step last?

62 degrees C, 1 minute

what is the temperature for the polymerization step and how long does it last?

72 degrees C, 1 minute

how many cycles do we run for this PCR experiment?

about 30 cycles

what is the temperature and how long does the final extension last during PCR?

72 degrees C, 5 minutes

a combination of the PCR primers CF and PR detect which allele?

purple allele

a combination of the PCR primers CF and NR detect which allele?

non-purple allele

what is SDS?

sodium dodecyl sulfate (detergent)