Quiz 1 Info

Question 1

For the project, we’re isolating … coding sequence (RNA –> …. –> …)

Answer 1

actin;
cDNA;
actin

Question 2

we’ll take actin and put it in a … - stable form that we can put into cells that will copy it for us
these cells have mechanisms to correct …

Answer 2

plasmid;

DNA replication mutations;

Question 3

plasmid will serve as … to copy actin sequence.

- Will then have … with … at the end - sequences of nucleotides recognized by restriction enzymes

Answer 3

PCR template;

ds actin; restriction sites

Question 4

plasmid will serve as PCR template to copy actin sequence.

• will then have ds actin with restriction sites at the end - sequences of nucleotides recognized by restriction enzymes
• will be cutting actin as well as … with the same restriction enzyme so that we get the same …
• put actin into …

Answer 4

GFP plasmid;
sticky ends;
GFP vector

Question 5

plasmid will serve as PCR template to copy actin sequence
- put actin into GFP vector:
Input this into cells and give it 24 hr to …
At that pt wherever we see … is where we have actin
Allows us to follow what actin is doing in …

Answer 5

start expression;
green;
live cells

Question 6

With using GFP plasmid, some of the … may not be there
working with … cells - everything is stuck in that configuration
- can use things like …, which gets rid of membranes and anything soluble gets washed away with it

Answer 6

localization sequences;
fixed;
methanol

Question 7

tools of fixation and localization - depends on …

can have … from fixed samples that show a perspective that isn’t actually what’s occurring within the cell

Answer 7

particular protein;

artifacts

Question 8

use … to get these fixed images of what protein is doing: fix cell and add the one that’s specific for protein of interest
…. binds to protein of interest

Answer 8

antibodies;

primary antibody

Question 9

…: one antibody that binds to protein of interest

- fluorophore on … region enabling us to see where that particular antibody has bound in the cell

Answer 9

direct immunofluorescence;

FC

Question 10

…: helps us to amplify the signal and make it easier to see where the protein is

• using …
• …: see primary antibody and each one of these has a fluorophore

Answer 10

indirect immunofluorescence;
multiple antibodies;
secondary antibody

Question 11

indirect immunofluorescence:
secondary antibody: increases signal in that particular location, making it easier to detect; a little more space between where that protein is and the … –> can get an …

Answer 11

fluorophore;

artifact

Question 12

immunofluorescence can only be done in …

- antibodies can take some time to make

Answer 12

fixed cells

Question 13

GFP folds on its own and forms its own …
… origin enables plasmid to copy itself when it’s in cells -> by having this, enables plasmid to be … and copied within the cell

Answer 13

chromophore;
pUC;
recognized

Question 14

has …, … resistance genes which enables us to use antibiotics to isolate cells with the plasmid

Answer 14

kandomycin; neomycin

Question 15

for this plasmid and that promote,r we don’t have a way to control expression in mammalian cells:
… promoter
could put … promoter on there (promoter from mammalian cells)
… promoters: add molecule that can then turn on expression only when you have the inducer

Answer 15

viral;
endogenous;
inducible

Question 16

… (…):
this is where we put in our gene sequence
has different … sequences, such that we can insert our gene sequence in the right frame and in the right direction

Answer 16

MCS;

multiple cloning site

Question 17

actin has area where it enables it to bind to … This enables it to … into filaments

Answer 17

ATP;

polymerize

Question 18

actin can only assemble in …
plus end and minus end relates to … and which end will be growing faster –> easier to add actin monomers onto … as compared to the other end

Answer 18

one direction;
shape;
plus end

Question 19

when assembled into filaments: … actin strands twisted together
plus ends of actin filaments will be at a …
minus ends will be going into the …

Answer 19

2;
membrane;
cytoplasm

Question 20

actin filaments also serve as tracks for …

most myosin motors move towards …

Answer 20

motor proteins; plus end

Question 21

Actin filaments:

• gives …
• creates little tracks within the cell for …/…
• …

Answer 21

structure;
movement/transport;
cell adhesion

Question 22

Actin filaments:

• at the leading edge of migrating cells, form …
• in …

Answer 22

dense network;

sarcomere

Question 23

Actin filaments:

- giving structure to cell surface through interactions with … –> helps with …, especially in …

Answer 23

spectrin;
flexibility;
RBCs

Question 24

Actin filaments:
- in kidneys and intestinal cells where there’s a lot of …, will form the … on the cell surface and help with increasing surface area to enhance absorption –> also forming interlocking network inside of these cells to help with …

Answer 24

filtration;
microvilli;
scaffolding

Question 25

actin’s sequence is …

• sequence is very similar between different organisms
• actin is also pretty …

actin has a … pattern to it -> helping to …

Answer 25

well-conserved;
universal;
honeycomb;
hold cells together

Question 26

actin is within … of cell

Answer 26

hollow

Question 27

easy to isolate expressed actin sequence because it’s pretty much present at every stage in …
- otherwise would need to know time point its expressed to …

Answer 27

early development;

isolate RNA sequence

Question 28

along with labeling protein of interest itself, can also label protein that … with protein of interest
don’t use genomic DNA due to presence of … - no sequences in plasmid that enable it to be …

Answer 28

interacts;
exons;
spliced together

Question 29

before we put plasmid into cell, we have to get … to ensure that it’s correct
for actin sequence, band that forms should be about … long

Answer 29

nucleotide sequence;

1142 bp;

Question 30

TOPO-TA cloning: uses …, which adds on extra … onto the 3’ end
- … linked onto ends of plasmid, acting as … to seal the backbone

Answer 30

tac thermase;
As;
topoisomerase;
ligase

Question 31

TOPO-TA cloning:
topoisomerase linked onto ends of plasmid, acting as ligase to seal the backbone –> gives us … that now has gene sequence in it
… plasmid
actin now in plasmid form
- has … sequences in it to enable the bacteria to make a ton of copies –> less likely to generate … due to the fact that bacteria is copying it

Answer 31

continuous plasmid;
TOPO;
replication;
mutations

Question 32

… cloning: using topo plasmid as template and use this in a PCR with primers specific to …
- primers will have additional sequence-introducing new sequence on the ends (… sequences)

Answer 32

directional cloning;
actin;
restriction site

Question 33

directional cloning:
primers will have additional sequence (restriction site sequences):
basing restriction sites on the ones we need to cut out …, don’t use ones that have sequences in … (want to avoid cutting into …)

Answer 33

GFP;
gene sequences;
coding sequence

Question 34

In MCS: restriction sites (about 20)
- compare these sites with those that aren’t found in … - these are the ones we’ll consider using

~
directional cloning product is gene sequence with … on end

Answer 34

actin sequence;

two restriction sites

Question 35

use restriction enzymes to cut in … and at ends of … enables actin to ligate into expression vector only in one direction
- want to make sure actin is going in same direction as … –> achieve this by having restriction sites at the end of the PCR product being …

Answer 35

MCS;
PCR product;
GFP;
different

Question 36

use restriction enzymes to cut in MCS and at ends of PCR product. Enables actin to ligate into expression vector only in one direction:

• stop codon must be further … from GFP
• controlling … and … to have continuous sequence going from GFP to gene of interest

Answer 36

downstream;
orientation;
reading frame