Quiz 2 PPTs Flashcards
What can go from RNA to DNA?
- …
- …
- …
retroviruses;
retroposons;
telomeres
For reverse transcription:
- Template: …
- primer: …, …, …
RNA;
oligo dTs; random hexamers; gene specific
For reverse transcription:
- … (aka …)
- …
RNA-dependent DNA polymerase; Reverse Transcriptase;
nucleotides
RT-PCR: ….
reverse transcription-polymerase chain reaction
RT-PCR:
- … RNA
- … RNA to generate …
isolate;
reverse-transcribe; cDNA
RT-PCR:
- cDNA becomes … for PCR to … specific sequence
- analyze RT-PCR products on …
template;
amplify;
agarose gels
RT-PCR:
- analyze RT-PCR products on agarose gels - note that this tells us … if the gene is …, but cannot ….
qualitatively;
expressed;
quantitate expression
4 steps of reverse transcription lab:
- …
- …
- …
- …
prep;
anneal;
extend;
inactivate
RT lab:
+RT is a …; reaction contains …
positive control;
all components
RT lab:
- RT: …, no …
- RNA: …, no …
negative control;
reverse transcriptase;
negative control;
RNA
RT lab:
- RT: Since no RT in reaction, no … can be made. Therefore, any product in PCR is due to …
cDNA;
gDNA contamination
RT lab:
- RNA: Since no RNA in reaction, nothing is there to … Therefore, any product in PCR is due to one of the … being …
provide a template;
reaction components;
contaminated
expected results for RT PCR:
+RT: …, …
- RT: …, … after pCR
- RNA: …, … after PCR
cDNA; product;
no cDNA; no product;
no cDNA; no product
RT lab:
… primers: a pair of primers that anneal to the sequence of the gene of interest and help … that sequence in PCR
gene specific;
copy
RT lab:
- Regarding primers, where to start depends on …
what it is that you want to do (what you want to copy)
RT lab:
- If you want to amplify a specific region, to make the primers, you need to first … that region and choose primers … that region that best fit the parameters
identify;
flanking
RT lab:
- forward primer: …
- reverse primer: …
direct sequence;
reverse and complement
RT lab:
PCR primer design:
- … to … nucleotides in length
…-…% GC content, melting temp … - … degrees C
18; 24;
40; 60;
45; 65
RT lab:
PCR primer design:
- …, …-… Gs or Cs at … end
GC clamp;
2-3; 3’
RT lab: primer secondary structure: - ... - ... - ... also want to avoid ... and ...
hairpins;
self dimers;
cross dimers;
runs; repeats
For RT-PCR, using … as template
single, isolated sequence
NanoDrop 2000:
this is the ….
- use …-… microliters of sample
- gives us …
spectrophotometer;
1; 2;
concentration
RT lab:
NanoDrop 2000:
gives us … and …
260/280;
260/230 ratios
The aborbance for a molar concentration of a substance with a path length of l cm determined at a …
Its value is obtained from the equation …
specific wavelength;
epsilon = A/cl
Beer lambert equation:
…
A = epsilon * b * c
Beer lambert: A = epsilon * b * c A = ... epsilon = ... b = ... c = ...
absorbance;
molar absorption coefficient;
path length;
concentration
RT lab:
260/280 ratio = …; DNA is …, RNA is …
“purity” of nucleic acids;
- 8;
- 0
RT lab:
260/230 ratio = second measure of …; range … - …
“purity”;
1.8 - 2.2
PCR lab:
- PCR = …
polymerase chain reaction
PCR lab:
PCR - rapid … of specific sequences of DNA
- bacteria vs. PCR - … vs …
amplification;
days; hours
PCR lab: 1983 - kary mullis - sickle cell anemia mutation; sanger sequencing --> ... + ... - later added ... - 1993 Nobel in chem
DNA polymerase; 2 oligo primers;
thermal cycling
PCR lab: PCR components: 1. ... 2. ... 3. ... 4. ... 5...
template; two primers; polymerase; nucleotides; thermocycler
PCR lab:
1. template: …, sequence in the template that is to be amplified = …
DNA;
target
PCR lab:
2. two primers: … whose sequence is … to …
oligonucleotides;
complementary;
ends of target sequence
PCR lab:
- polymerase: …
- thermocycler: originally done by transferring between …
DNA-dependent DNA polymerase;
water baths
PCR lab:
cycling:
… steps per cycle, …-… cycles
three;
25-35
PCR lab: cycling steps: 1. ... 2. ... 3. ...
denature;
anneal;
elongate
