Quiz 3 Info

Question 1

We did the Topo Ta cloning to give actin sequence … (??) so that we can then insert actin into the …

Answer 1

restriction sites (not sure if this is even correct);
gfp plasmids

Question 3

PCR on bacteria from colonies. Touching pipet to colony of plate and putting that in PCR

• cycles of heating and thawing will … and …, such that it can then be used as template for PCR
• If we were to do this, we would still have to …

Answer 3

break bacteria; release plasmid;

culture the right colony

Question 4

EcoR1 and Sac2 restriction sites in … and … primers for actin, respectively
To design primers, had to see what restriction sites are in … but not in … so that actin coding sequence can be flanked by those restriction sites

Answer 4

forward; reverse;
MCS;
actin sequence

Question 5

have to … gfp plasmid by cutting it with …

- cutting it with ecor1 and sac2 as well to generate …

Answer 5

linearize;
restriction enzymes;
same sticky ends

Question 6

ecor1 and sac2 will always cut at the same restriction site so long as … aren’t wildly off

• for ecor1, cuts between … and …
• for sac2, cuts between last … and … when it sees its particular restriction site

Answer 6

salt concentrations;
g; a;
c; g

Question 7

we ran out plasmids against 1 kb ladder - technically not the right control for plasmids bc 1 kb ladder is made of … fragments of DNA which will migrate differently than supercoiled/nicked DNA.
- nicked will move … and supercoiled will move …. in gel and will move as though it’s … than it really is

Answer 7

linear;
larger than apparent size;
faster;
smaller

Question 8

if pcr 2.1 vector just seals back on itself, it will be … bp

• supercoiled will be migrating such that it’s just over the … band
• if they have actin sequence, supercoiled will be migrating almost as far as … bp band in the ladder
• actual size is …

Answer 8

3929;
2000 bp;
3054;
5071

Question 9

pcr 2.1 vector:
better control would be to use … in one of the lanes and if our plasmid doesn’t migrate as far, it would indicate that we have …

Answer 9

blue colony; DNA

Question 10

Did M13 to take a look at what might be … in the plasmids

• plasmid has m13 sequences on it that are about … bp apart - if no insert, will get product at …
• if insert is actin - we’re adding … bp such that the total size is … bp

Answer 10

inserted; 
200;
200 bp; 
1142; 
1343

Question 11

… that actin was inserted didn’t matter when making too plasmid

• now it matters when inserting into gap plasmid
• has to be in same … as gfp before it, as well

Answer 11

direction;

reading frame

Question 12

this time, we’re cloning into a … plasmid - have to … first, because right now it can’t take up any DNA

Answer 12

closed;

cut into them

Question 13

cloning into a closed plasmid:
… comes after gfp coding sequence, which is after … sequence
- we cut the plasmid by using … cutting into …

Answer 13

multi cloning site; 
GFP coding; 
promoter; 
restriction enzymes; 
restriction sites

Question 14

use restriction enzymes to cut plasmid:

- cutting ends of actin so it has … Cut gfp plasmid with the … so it has compatible … to it

Answer 14

specific overhangs;
same restriction enzymes;
overhangs

Question 15

Nucleases:

• break … in nucleic acid
• substrate depends on …

Answer 15

phosphodiester bonds;

enzyme itself

Question 16

Nucleases:

• substrate depends on enzyme itself:
• … - break down RNA
• … - break down DNA
• … - only cut at the ends
• … - cut in the middle of a sequence

Answer 16

ribonuclease;
deoxyribonuclease;
exonuclease;
endonucleases

Question 17

Nucleases:

- endonucleases: we’re using …, which binds to the particular sequence of nucleotides and cuts …

Answer 17

type 2;

inside of that sequence

Question 18

Nucleases:
we’re using type 2 endonucleases:
- … - cut on …
- their restriction sites are … - if you read them … on either strand, they will have same sequence

Answer 18

dimers;
both strands;
palindromes;
5’ to 3’

Question 19

restriction endonucleases evolved to protect bacteria from … so that if there was any linear DNA that entered, the endonucleases that were endogenous to the bacteria could attack and … that was infecting the cell
- bacteria protects its own DNA by … enzymes that recognize the same sequences as restriction enzymes, but add … on DNA so that restriction enzymes don’t cut it

Answer 19

viruses;
break down that DNA;
modification;
methyl groups;

Question 20

restriction site = …

• for type 2, they are the same because enzymes bind and cut …
• for the ones we’re using, they are … nucleotides long
• … = palindrome

Answer 20

recognition site;
within the same sequence;
6;
inverted repeat

Question 21

binding and cutting is very specific as long as … are right
- how often DNA will be cut depends on how often … is in DNA –> probability is higher for … restriction site sequences

Answer 21

salt cones;
restriction site sequence;
shorter

Question 22

use restriction enzymes for:

• …
• …

Answer 22

restriction mapping;

cloning

Question 23

Use restriction enzymes for:
- restriction mapping: comparing two pools of DNA by cutting them with … –> if the DNA is the same, will get … Otherwise, will get …

Answer 23

the same enzymes;
same fragments;
different fragment lengths

Question 24

we’re cutting gfp and actin pcr product so that we can …

Answer 24

ligate them together

Question 25

using … primers that also have …

- we’re adding … onto forward and reverse primer

Answer 25

gene specific;
restriction site sequence;
restriction site;

Question 26

using gene specific primers that also have restriction site sequence:
we’re adding restriction sites onto forward and reverse primer:
- … restriction on forward
- … restriction on reverse
^ this is to ensure that actin will …

Answer 26

upstream;
downstream;
go in the right direction

Question 27

using gene specific primers that also have restriction site sequence:
we’re adding restriction sites onto forward and reverse primer:
- have an additional nucleotide to ensure it’s in the …

Answer 27

right reading frame