LAB 3 Flashcards
SYBR Green I dye
binds dsDNA in minor groove
great sensitivity
unbound=little fluorescence
bound to dsDNA during annealing and elongation = fluorescence increases 1000 fold
denaturation = ssDNA, little fluorescence
fluorescence measurements taken at the end of the elongation phase (max dye binding)
what is the threshold cycle
Ct
cycle at which fluorescence is determined to be statistically significant above background
inversely proportional to the log of the initial number of molecules
from this we can quantify the initial template number
RNA isolation
what do you get if you run all the RNA on a gel electrophoresis
three predominant bands: rRNA (28S, 18S and 5S) and some tRNA
how do you calculate the purity of your isolation
what do you need to do to your RNA before you do qPCR
need to make RNA into cDNA using reverse transcriptase (viral enzyme) and oligoT primers
how is primer specificity/efficiency determined with qPCR?
lab 3 week 1 protocol
isolate and purify RNA from HCT116 colorectal cancer cells, 5-FU treated or not
quantify RNA spectrophotometrically and analyse quality on agarose gel
perform cDNA synthesis using RT to make mRNA into DNA
isolate the RNA
wash it with ethanol
what is the challenge with isolation of RNA and how they are overcome
RNA is much less stable than DNA
high temp or high pH can denature RNA
RNase enzymes are also a concern
RNase are ubiquitous and very stable
controlled by using chaotropic lysis buffers (such as guanidinium thiocyanate) that completely disrupt non covalent interactions and denature RNases
what is the beer lambert equation and the extinction coefficient or RNA at 260nm
A260=epsilon c l
25µL/µg/cm
lab 3 week 2 protocol
end point and real time PCR on the cDNA of p21 to analyse its expression normalised to the housekeeping gene GAPDH
for endpoint PCR:
create master mizes
prepare 1/5 dilution of each cDNA
load p21 and GAPDH on same gel to compare intensities
for qPCR:
master mix for p21 and GAPDH
prepare 1/80 dilution of cDNA
how is primer efficiency determined (from the dilutions)
how is fold induction/fold change calculated
imageJ analysis of intensities
divide normalised p21 intensity for treated cells by the normalised p21 intensity of untreated cells
how should the fold induction compare between wild type and null p53 cells?
p53 mutant cells resist 5-FU induction and are not able to activate p21, and therefore the change in gene expression is not as large as for wild type p53 cells
what is the Pfaffl equation for calculating the relative change in nucleic acid between a control and a treatment sample (from qPCR data)
isolation of RNA protocol
first, find the Ct values by looking at where the gene crosses the threshold
15 for red
26 for green
how does a northern blot work
how does an RNAase protection assay work
