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Biotech > Quiz 2 Info - 3 > Flashcards

Quiz 2 Info - 3 Flashcards

1
Q

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

A

bend

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2
Q

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

A

slower;
2 days;
4 hrs

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3
Q

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

A

e. coli;

intestines

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4
Q

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 …
A

gDNA;
the same;
a lot quickly

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5
Q

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

A

37;
4;
suspended animation

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6
Q

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 …

A

growing; dividing;

killed them off

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7
Q

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

A

other bacteria;

contaminate

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8
Q

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

A

circular;

dsDNA

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9
Q

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

A

non-bacterial;

easily degraded

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10
Q

can use e. coli for:

  • …
  • …
  • …
  • produce …
A

cloning;
libraries;
bioremediation;
recombinant proteins

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11
Q

can use e. coli for:

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

A

ds;

plasmids

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12
Q

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
A

screen;
DNA;
specific DNA sequences

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13
Q

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 …
A

environmental disasters;
conformation;
function

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14
Q

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)

A

protein modifications;
disulfide bridges;
glycosylate

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15
Q
  • e coli developed from strain with … knocked out of it to ensure that it can’t …
A

virulent strains;

infect researchers

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16
Q
  • 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 …
A

grow;
on the plate;
leucine

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17
Q
  • competent bacterial cells - just means that they are able to …
  • electrocompetent cells are … first
A

take up DNA;

electrocuted

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18
Q
  • electrocompetent cells are grown … at … temps. then resuspended in … rather than …
A

slowly;
lower;
glycerol; salts

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19
Q
  • electrocompetent: get DNA into them by … to open up … in the membranes such that DNA is … - it … in the process
A

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

20
Q

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

A

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

21
Q

chemically competent:

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

A

take up more DNA;

recover

22
Q

chemically competent:

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

A

fragile;
vortex;
vigorously

23
Q

chemically competent:

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

A

heat shock;
recovery;
fix membranes

24
Q

chemically competent:

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

A

efficient

25
Q

prior to topo-ta cloning, we have a … plasmid:

  • has …
  • has …
  • has … and … resistance genes
A

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

26
Q

prior to topo-ta cloning, we have a linearized plasmid:

- before we do transformation, we’re going to … the ends to make it …

A

ligate;

circular

27
Q

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
A

LB amp;

ampicillin

28
Q

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 …
A

beta lactamase;

make cell wall

29
Q

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

A

ring structure;

inhibit

30
Q

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

A

transferases;

block translation

31
Q

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

A

channel;

tetracycline

32
Q

TOPO-TA cloning:

- starts with pcr product made by …, which adds an extra … to the … ends of the strands it makes

A

taq polymerase;

a; 3’

33
Q

TOPO-TA cloning:

- ta cloning takes advantage of extra as - put into plasmid that has … that As can base pair with

A

T overhangs

34
Q

TOPO-TA cloning:

- linear plasmid with T overhangs also has … joined to each end of that plasmid –> …

A

enzyme;

topoisomerase

35
Q

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
A

dna replication;
strain;
ligation

36
Q

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 …

A

seals the backbone;
takes longer;
lower temperature

37
Q

TOPO-TA cloning:

- cell … has topoisomerase

A

naturally

38
Q

TOPO-TA cloning:

- …, … can insert themselves into topo plasmid and sometimes plasmid just …

A

individual nucleotides;
primers;
closes on its own

39
Q

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 …
A

actin;

active in solution

40
Q

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 …
A

pipet;
high;
equal

41
Q

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
A

1 kb;
low mass;
interacting with one another

42
Q

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

A

breaking off As

43
Q

heat shock is very … sensitive

recovery media can just be …

A

time sensitive;

LB

44
Q

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

A

have plasmid;

ligate back on itself

45
Q

x gal:

  • colonies that are cream/white color have …
  • blue colonies are …
A

something inserted in plasmid;

just the plasmid

46
Q

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

A

lac z alpha;

beta galactosidase

47
Q

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
A

disrupts;
still intact;
cleaves;

Biotech (17 decks)

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