Quiz 2 Info - 2

Question 1

start with urchin RNA then do reverse transcription to form … then do PCR to form … containing ….

Answer 1

single stranded cDNAs;
dsDNA;
actin sequence

Question 2

… that enables transcription of the following sequence in mammalian cells
… will be template for PCR
we sequence after PCR to ensure that …

Answer 2

CMV viral promoter;
RT results;
no mutations were introduced

Question 3

in pcr:

- heat up sample to 95 degrees to … into … Polymerase can then … and primers can …

Answer 3

denature dsDNA;
ssDNA;
bind;
bind

Question 4

in pcr:

- forward primer binding and containing …

Answer 4

start codon

Question 5

in pcr:

• cool it down for primers and polymerase to …
• warm back up to 70 degrees such that polymerase is … and can …

Answer 5

anneal;
active;
add nucleotides to 3’ end

Question 6

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

Answer 6

template;
sequence of primers;
52

Question 7

in pcr:

• done for …-… cycles
• major product has … sequence on one side and … sequence on other side –> …

Answer 7

25; 35;
forward primer;
reverse primer;
dsDNA

Question 8

in pcr:

• our primers start at …
• reverse primer is at …
• Thus, length of our sequence is approximately …

Answer 8

ATG;
1141 bp;
1141 bp

Question 9

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 …

Answer 9

genomic DNA;

introns

Question 10

in pcr:

- before cycling, 2 min … for …

Answer 10

hot start;

denaturing

Question 11

in pcr:

• before cycling, 2 min hot start for denaturing –> helps with … and helps prevent primers from …
• this is along with getting rid of …

Answer 11

activating polymerase;
sticking to where they shouldn’t be;
double stranded

Question 12

in pcr:

- melting temp: … point where you have … and …

Answer 12

inflection;
half of your primers bound;
half unbound

Question 13

in pcr:

- melting temp: half of DNA is … and half is …

Answer 13

ds;

ss

Question 14

in pcr:

• melting temp is based on … in the primer
• equation for melting temp: …

Answer 14

nucleotides; 
4 deg(G + C) + 2 deg(A + T)

Question 15

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 …

Answer 15

both primers;
drop it;
bind

Question 16

in pcr:

- for elongation time: …/… s for every …-… bp

Answer 16

30; 60;

500; 1000

Question 17

in pcr:

for elongation time for actin, about … –> but … should be sufficient

Answer 17

2 min;

1 min

Question 18

in pcr:

- using taq polymerase –> can withstand …

Answer 18

fluctuations in temp

Question 19

in pcr:

- gotaq master mix combines …, …, … and …

Answer 19

buffer;
dNTPs;
MgCl2;
polymerase

Question 20

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

Answer 20

slower;
reduce errors;
green dye

Question 21

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

Answer 21

correct temperature;
evaporates; condenses;
run down;

Question 22

in pcr:

- can use pcr to… into sequence (i.e. …) –> putting them into …

Answer 22

put mutations;
mutagenesis;
primer