Quiz 1 Info Flashcards by Gail I

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

actin;
cDNA;
actin

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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 …

plasmid;

DNA replication mutations;

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plasmid will serve as … to copy actin sequence.

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

PCR template;

ds actin; restriction sites

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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 …

GFP plasmid;
sticky ends;
GFP vector

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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 …

start expression;
green;
live cells

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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

localization sequences;
fixed;
methanol

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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

particular protein;

artifacts

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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

antibodies;

primary antibody

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…: one antibody that binds to protein of interest

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

direct immunofluorescence;

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…: 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

indirect immunofluorescence;
multiple antibodies;
secondary antibody

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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 …

fluorophore;

artifact

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immunofluorescence can only be done in …

- antibodies can take some time to make

fixed cells

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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

chromophore;
pUC;
recognized

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

kandomycin; neomycin

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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

viral;
endogenous;
inducible

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… (…):
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

MCS;

multiple cloning site

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

ATP;

polymerize

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

one direction;
shape;
plus end

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

2;
membrane;
cytoplasm

actin filaments also serve as tracks for …

most myosin motors move towards …

motor proteins; plus end

Actin filaments:

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

structure;
movement/transport;
cell adhesion

Actin filaments:

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

dense network;

sarcomere

Actin filaments:

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

spectrin;
flexibility;
RBCs

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 …

filtration;
microvilli;
scaffolding

actin's sequence is ... - sequence is very similar between different organisms - actin is also pretty ... actin has a ... pattern to it -> helping to ...

well-conserved; universal; honeycomb; hold cells together

actin is within ... of cell

hollow

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 ...

early development; | isolate RNA sequence

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 ...

interacts; exons; spliced together

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

nucleotide sequence; | 1142 bp;

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

tac thermase; As; topoisomerase; ligase

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

continuous plasmid; TOPO; replication; mutations

... 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)

directional cloning; actin; restriction site

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 ...)

GFP; gene sequences; coding sequence

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

actin sequence; | two restriction sites

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 ...

MCS; PCR product; GFP; different

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

downstream; orientation; reading frame