PV92 Flashcards
biorad manual
PCR uses heat-resistant Taq polymerase from?
Thermus aquaticus
biorad manual
volume of saline to be transfer in the micro test tube.
how long will u rinse ur mouth using saline?
1 ml
30 seconds
sample of choice
- hair follicles
- oral epithelial cells (cheek cells)
- drop of blood
centrifuge time for saline
what u should see after centrifugation?
2 minutes
match-head sized pellet of whitish cells at the BOTTOM of the tube.
biorad manual
temp where it loosens connective tissue and inactivates DNAses
hint: temp of water bath
56C
biorad manual
ruptures cell membranes and denatures protein
this is the temp of?
100C
dry bath
discussion
PV92 PCR is to detect — which composed of — nucleotides
to detect alu transposon
composed of 300 nucleotides
discussion
marker of human migration
what is the purpose?
Alu I restriction enzyme
relatedness among individuals
discussion
locus/region of DNA is called?
PV92
discussion
GIVE THE GENOTYPE AND SIZE OF PCR PRODUCTS
- (+/+)
- (-/-)
- (+/-)
- (+/+) = homozygous 941 bp
- (-/-) = homozygous 641 bp
- (+/-) = heterozygous 941 and 641 bp
discussion
what is the purpose of instagene matrix?
to lyse cheek cells
discussion
after adding instagene matrix, what’s the next step?
dry bath 100C for 5 mins
discussion
contents of master mix (6)
- DNA pol (Taq polymerase)
- magnesium ion (enzyme cofactor)
- PCR primer
- salt buffer
- template
- nucleotides (dATP, dCTP, dGTP, dTTP)
discussion
why need control?
to check the reagent
discussion
regulates temperature of the sample in thermal cycler
aluminum blocks
discussion
used for staining of agarose gel
orange G stain
discussion
how do you prepare gel casting tray?
- comb to create wells
- buffer
- set the volts
discussion
how do you report?
- homozygous
- homozygous (-)
- heterozygous
can be positive/negative
- homozygous = mm
- homozygous (-) = mn
- heterozygous = nn
discussion
enumerate the steps in genomic DNA protocol
separate in each phase
SAMPLE PREP
1. sample (if oral, use saline mouth wash)
2. cell lysis
3. centrifuge to separate the cells
3. add instagene matrix to lyse check cells
4. dry bath 100C for 5 mins
5. water bath 56C for 10 mins
REAGENT PREP
1. master mix prep
PCR AMPLIFICATION/ANALYSIS
1. denaturation
2. annealing
3. extension
4. gel electrophoresis
5. stain the gel
6. visualize the gel under UV
should have a band that indicates PCR was successful.
discussion
enumerate the steps in gel electrophoresis
what’s the complete name of the electrophoresis we used?
- prepare agarose
- prepare gel cating tray
- electrophoresis prep
- electrophoresis loading
- staining of agarose gel
agarose gel electrophoresis
discussion
principle stating that the genetic variation in a population will remain constant from one generation to the nex in the absence of distrurbing factors.
Hardy-Weinberg Equilibrium
p^2 + 2pq + q^2 = 1
discussion
how to calculate genotypic frequencies?
number of category / total number
discussion
negatively charged, microscopic beads that chelate or grab, metal ions out of solution. traps metal ions, such as Mg2+ which are required as catalysts or cofactors in enzymatic reactions
but what is the function?
instagene matrix
binds cellular magnesium ions
biorad manual
PV92 is used in?
- population genetics
- paternity analysis
- forensics
lab manual
temp for each step of PCR
- denaturation = 94-98C
- annealing = 50-65C
- extension = 72-75C
remember! should repeat 20-40 cycles
biorad manual
PV92 locus is on chromosome?
chromosome 16
biorad manual
approx. — Alu copies per haploid genome, representing about — of the genome
500,000 Alu copies per haploid, representing about 5% of the genom
biorad manual
genotypes
- +/-
- -/-
- +/-
biorad manual
determionation and analysis of alu frequency in a population
enumerate step-by-step
- amplify Alu insert from represenative sample population
- calculate the expected allelic and genotypic frequencies
- perform chi-square test
