Quiz 2 Info - 3 Flashcards
nucleotide mismatch can be recognized by bacteria bc it causes DNA to …
bend
using bacteria to replicate our bacteria is … - takes about … whereas PCR takes about …
slower;
2 days;
4 hrs
we’re using … to clone the plasmid. this is initially isolated from …
e. coli;
intestines
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
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
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
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
we’re putting in … pieces of … into the bacteria
circular;
dsDNA
linear pieces are immediately recognized as … and will be … by the bacteria
non-bacterial;
easily degraded
can use e. coli for:
- …
- …
- …
- produce …
cloning;
libraries;
bioremediation;
recombinant proteins
can use e. coli for:
- libraries: making RNA … and put it into … that are then put in the bacteria
ds;
plasmids
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
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
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
- e coli developed from strain with … knocked out of it to ensure that it can’t …
virulent strains;
infect researchers
- 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
- competent bacterial cells - just means that they are able to …
- electrocompetent cells are … first
take up DNA;
electrocuted
- electrocompetent cells are grown … at … temps. then resuspended in … rather than …
slowly;
lower;
glycerol; salts
- 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;
