Quiz 2 Info - 2 Flashcards
start with urchin RNA then do reverse transcription to form … then do PCR to form … containing ….
single stranded cDNAs;
dsDNA;
actin sequence
… that enables transcription of the following sequence in mammalian cells
… will be template for PCR
we sequence after PCR to ensure that …
CMV viral promoter;
RT results;
no mutations were introduced
in pcr:
- heat up sample to 95 degrees to … into … Polymerase can then … and primers can …
denature dsDNA;
ssDNA;
bind;
bind
in pcr:
- forward primer binding and containing …
start codon
in pcr:
- cool it down for primers and polymerase to …
- warm back up to 70 degrees such that polymerase is … and can …
anneal;
active;
add nucleotides to 3’ end
in pcr:
- the products of first round will be … for next round allowing for further amplification
- range of temps for annealing is dependent on … themselves –> for us its … degrees
template;
sequence of primers;
52
in pcr:
- done for …-… cycles
- major product has … sequence on one side and … sequence on other side –> …
25; 35;
forward primer;
reverse primer;
dsDNA
in pcr:
- our primers start at …
- reverse primer is at …
- Thus, length of our sequence is approximately …
ATG;
1141 bp;
1141 bp
in pcr:
- length of sequence is approximately 1141 bp –> when we do PCR and run product on gel, we expect to see a band at that size
- if we have anything bigger, … was used as a template and the product contains …
genomic DNA;
introns
in pcr:
- before cycling, 2 min … for …
hot start;
denaturing
in pcr:
- before cycling, 2 min hot start for denaturing –> helps with … and helps prevent primers from …
- this is along with getting rid of …
activating polymerase;
sticking to where they shouldn’t be;
double stranded
in pcr:
- melting temp: … point where you have … and …
inflection;
half of your primers bound;
half unbound
in pcr:
- melting temp: half of DNA is … and half is …
ds;
ss
in pcr:
- melting temp is based on … in the primer
- equation for melting temp: …
nucleotides; 4 deg(G + C) + 2 deg(A + T)
in pcr:
- get melting temp for … and then … a couple degrees to get annealing temp –> this is to push reaction to where more is able to …
both primers;
drop it;
bind
in pcr:
- for elongation time: …/… s for every …-… bp
30; 60;
500; 1000
in pcr:
for elongation time for actin, about … –> but … should be sufficient
2 min;
1 min
in pcr:
- using taq polymerase –> can withstand …
fluctuations in temp
in pcr:
- gotaq master mix combines …, …, … and …
buffer;
dNTPs;
MgCl2;
polymerase
in pcr:
- gotaq master mix: if you increase MgCl2, it makes enzyme work … but it should help …
- … in gotaq allows it to be immediately loaded onto gel
slower;
reduce errors;
green dye
in pcr:
- use tubes with thin walls for PCR so that reagent inside can have … as we change it
- domed cap so that if anything … and … in the domed cap, it will … side of tube and go back to the rest of the sample
correct temperature;
evaporates; condenses;
run down;
in pcr:
- can use pcr to… into sequence (i.e. …) –> putting them into …
put mutations;
mutagenesis;
primer
