Quiz 2 Info - 3 Flashcards by Gail I

nucleotide mismatch can be recognized by bacteria bc it causes DNA to …

bend

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using bacteria to replicate our bacteria is … - takes about … whereas PCR takes about …

slower;
2 days;
4 hrs

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we’re using … to clone the plasmid. this is initially isolated from …

e. coli;

intestines

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we’re using e. coli to clone the plasmid:

initially isolated from intestines
… won’t be interacting with plasmids
if you work with a single colony, all the cells within that colony are …
can get …

gDNA;
the same;
a lot quickly

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we’re using E coli to clone the plasmid:

- e coli likes to be at … degrees C but can store at … degrees C to help with keeping cells in almost a … state

37;
4;
suspended animation

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we’re using E coli to clone the plasmid:
- e coli likes to be at 37 degrees C but can store at 4 degrees C to help with keeping cells in almost a suspended animation state - aren’t actively … and …, but we haven’t …

growing; dividing;

killed them off

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we’re using E coli to clone the plasmid:
- e coli likes to be at 37 degrees C but can store at 4 degrees C to help with keeping cells in almost a suspended animation state –> also makes it less likely that … will … the plate

other bacteria;

contaminate

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we’re putting in … pieces of … into the bacteria

circular;

dsDNA

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linear pieces are immediately recognized as … and will be … by the bacteria

non-bacterial;

easily degraded

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can use e. coli for:

…
…
…
produce …

cloning;
libraries;
bioremediation;
recombinant proteins

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can use e. coli for:

- libraries: making RNA … and put it into … that are then put in the bacteria

ds;

plasmids

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can use e. coli for:

libraries - could then … bacteria to see which has the sequence we want
libraries to hold sequences in … form in an easily accessible way that enables us to pull out … easily

screen;
DNA;
specific DNA sequences

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can use e. coli for:

bioremediation: cleaning up …
produce recombinant proteins: if you make too much protein too quickly it won’t have the right … and will therefore not have the right …

environmental disasters;
conformation;
function

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can use e. coli for:
- produce recombinant proteins: also, it’s a prokaryotic system so we won’t have the same … that we see in eukaryotes (e.g. can’t form …, can’t …, etc)

protein modifications;
disulfide bridges;
glycosylate

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e coli developed from strain with … knocked out of it to ensure that it can’t …

virulent strains;

infect researchers

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tried to make an e coli strain that can’t colonize within people - but initial strain didn’t … very well
engineered such that it can only grow … bc it needs …

grow;
on the plate;
leucine

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competent bacterial cells - just means that they are able to …
electrocompetent cells are … first

take up DNA;

electrocuted

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electrocompetent cells are grown … at … temps. then resuspended in … rather than …

slowly;
lower;
glycerol; salts

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electrocompetent: get DNA into them by … to open up … in the membranes such that DNA is … - it … in the process

shocking them w/ electricity;
holes;
sucked in;
kills off half the cells

chemically competent - grown …, harvested and then resuspended in solution with high concentration of … which …

slowly;
salts;
damages the membrane (pokes holes into membrane0

chemically competent:

- can incubate cells with plasmid and then heat shock them - helps cells …, and we allow them to …

take up more DNA;

recover

chemically competent:

- they are very … bc of the holes (so we can’t do things like …). Can’t pipet …

fragile;
vortex;
vigorously

chemically competent:

- if the cells are exposed to warm temps - …, they then start their … process to …

heat shock;
recovery;
fix membranes

chemically competent:

- not as … as electrocompetent, but we have a lot of colonies to work with

efficient

prior to topo-ta cloning, we have a ... plasmid: - has ... - has ... - has ... and ... resistance genes

linearized; selection marker; origin of replication; ampicillin; kanamycin

prior to topo-ta cloning, we have a linearized plasmid: | - before we do transformation, we're going to ... the ends to make it ...

ligate; | circular

not all of the bacteria will take up plasmid and we don't want all of them to grow - we only want bacteria with plasmid to grow - to control this, we put it on ... plates that contain ... --> thus, only bacteria with plasmids will grow colonies

LB amp; | ampicillin

not all of the bacteria will take up plasmid and we don't want all of them to grow - use LB amp plates. - plasmid enables bacteria to make ... which is what allows it to live in the presence of antibiotic - ampicillin interferes with enzymes that ...

beta lactamase; | make cell wall

not all of the bacteria will take up plasmid and we don't want all of them to grow - beta lactamase cuts ... structure in ampicillin such that it can't ... enzymes that make up the cell wall anymore

ring structure; | inhibit

resistance genes to chloro, strepto and kana are ... that put functional groups on those groups (?) such that they can't ... anymore

transferases; | block translation

tetracycline resistance gene is a ... that pumps ... out of the cell

channel; | tetracycline

TOPO-TA cloning: | - starts with pcr product made by ..., which adds an extra ... to the ... ends of the strands it makes

taq polymerase; | a; 3'

TOPO-TA cloning: | - ta cloning takes advantage of extra as - put into plasmid that has ... that As can base pair with

T overhangs

TOPO-TA cloning: | - linear plasmid with T overhangs also has ... joined to each end of that plasmid --> ...

enzyme; | topoisomerase

TOPO-TA cloning: - topoisomers: involved in ... (ahead of replication fork to relieve ... on twisted DNA) - we use the ... part that topoisomerase does and using that to make an intact circular plasmid

dna replication; strain; ligation

TOPO-TA cloning: mixing pcr product with A overhangs with linear plasmid with T overhangs: - base pairing between a and t occurs - topoisomerase then ... so that we have a continuous piece of dsDNA - when using ligase instead of topo, it ... and have to do it at ...

seals the backbone; takes longer; lower temperature

TOPO-TA cloning: | - cell ... has topoisomerase

naturally

TOPO-TA cloning: | - ..., ... can insert themselves into topo plasmid and sometimes plasmid just ...

individual nucleotides; primers; closes on its own

individual nucleotides, primers can insert themselves into topo plasmid. sometimes plasmid just closes on its own - not necessary to have actin there for the plasmid to close - have to try to select for bacteria that not only took up plasmid, but that specifically have ... - have to start transformation within 5 mins of cloning bc topoisomerase is still ...

actin; | active in solution

for rt pcr, we wanted ot have a single band of 1141 bp only in +RT lane - that would allow us to just ... out of PCR sample for topo ta cloning - if pcr product is a huge bright band, the likelihood that actin sequence would be incorporated in plasmid is ... If same brightness as, for example, primers, likelihood of actin and primers being incorporated is essentially ...

pipet; high; equal

for rt pcr: - we used a ... ladder and a ... ladder (this one had a 1200 bp band) - in our results, primers were ... forming a banding pattern below 1000 bp

1 kb; low mass; interacting with one another

if we cut out and purify from pcr gel to obtain the actin sequence, there's a risk of ... for ligation reaction

breaking off As

heat shock is very ... sensitive | recovery media can just be ...

time sensitive; | LB

x gal enables us to select between colonies that ... vs those that just had plasmid ...

have plasmid; | ligate back on itself

x gal: - colonies that are cream/white color have ... - blue colonies are ...

something inserted in plasmid; | just the plasmid

we are cloning into ... region on our plasmid - this is the alpha subunit of ...

lac z alpha; | beta galactosidase

we are cloning into lac z alpha region on our plasmid: - these cells only have part of beta galacto enzyme and they themselves can't cut x-gal. when cloning into that plasmid, we're cloning into that alpha subunit. if anything gets cloned into plasmid, it ... that sequence - if plasmid closes on itself, alpha subunit sequence is ... and it finds the rest of the mutant beta galacto being made by the cells themselves, takes x gal and ... it to make blue dye on the plate --> this is why blue colonies mean no insert into plasmid

disrupts; still intact; cleaves;