Quiz 1 Info Contd 2 Flashcards
Flame creates … that draws things up and away from your plates - helps make sure that nothing … plates/cultures that you’re working with
convection currents;
contaminates
DNA:
- …
- … products
- …
- …
- …
- … = …/… –> copy of RNA that’s been isolated through use of …
plasmids; PCR; digests; pEGFP; TOPO*TA; cDNA; copy DNA; complementary DNA; reverse transcriptase
RNA:
- … (already isolated from different stages of development, eggs, 6 hrs, 18 hrs, 23 hrs - when cells migrate into the … of the embryos, 72 hrs - … stage, free swimming, look like easel, fully functioning gut, have almost a skeleton that gives them some … and enables them to maintain their form)
sp RNA (sp is the sea urchin);
inner cavity;
pluteus;
shape
RNA:
- sp RNA –> using … - has …, …, … and …
total RNA; mRNA; tRNA; rRNA; small RNAs
takes about …-… years for urchins to be fully developed
urchins develop …, so you can see what’s happening at each stage - can also get a lot of samples to work with
1.5; 2;
externally
urchins develop externally, so you can see what’s happening at each stage
can also get a lot of samples to work with
- if you fertilize samples in mass, they develop along … (go from fertilization to first division 1.5 hrs later) –. will be at the same point of …, enables you to obtain …
the same timescale;
development;
large quantities
urchins develop externally, can get a lot of samples to work with
if you fertilize samples in mass, they develop along the same timescale:
- can collect …/… from those particular stages
- for …, can fix and stain them
proteins/RNA;
immunolocalization
early stages of development for urchins are very similar to early … development - proteins behaving the same way
RNA can form … - complementary binding to prevent attack by …
human development;
secondary structure;
RNAses
RNA tends to be more … and … compared to DNA
transient;
unstable
RNA tends to be more transient and unstable compared to DNA:
… group on … enables RNAses to attack and break the bond –> catalyzes … attack, which can also occur in the absence of …
hydroxyl;
2’;
2’ OH;
RNAse
… also contributes to RNA instability
- it doesn’t have the … group that’s on …
uracil;
methyl;
thymine
uracil also contributes to RNA instability
- … in DNA helps with protecting DNA, makes it no longer recognizable to … that can cut it, helps to sequester DNA and prevent …
methyl;
restriction enzymes;
transcription
uracil also contributes to RNA instability:
- in RNA, cytosine can be … and become uracil. If that occurs, particularly with mRNA, that can change what gets …. in the amino acid sequence. This is not too bad though bc RNA is … in the cell so there wouldn’t be that much crap protein formed
deaminated;
coded;
transient
small RNAs regulate whether mRNA gets … or …
… is a form of regulation
translated;
degraded;
transience
RNA isolation similar to DNA isolation, but some tweaks due to the fact that it’s less stable.
since RNA is susceptible to RNAses in cell and environment, it has to get isolated in the presence of a … (…)
strong denaturant;
trisol
since RNA is susceptible to RNAses in cell and environment, it has to get isolated in the presence of a strong denaturant (trisol)
- using denaturant to denature any …, specifically RNAses, that are present
- we then remove proteins - take … phase of trisol that has RNA, add … to get RNA from that phase
proteins;
aqueous;
ethanol
Since RNA is susceptible to RNAses in cell and environment, it has to get isolated in the presence of a strong denaturant
- we then remove proteins. take aqueous phase of trisol that has RNA, add ethanol to get RNA from aqueous phase –> causes …; after this occurs, …, remove …, ….
RNA precipitation;
centrifuge;
ethanol;
resuspend in water
Since RNA is susceptible to RNAses in cell and environment, it has to get isolated in the presence of a strong denaturant
- we resuspend in water, using … treated water –> this water has 3 different … groups on it that helps with …
DEPC;
amine groups;
inactivating RNAses
Since RNA is susceptible to RNAses in cell and environment, it has to get isolated in the presence of a strong denaturant
- after isolation, we have all the RNAs that were present within the cell –> if we were to run on gel, would see …(…), … bands (…) and a smear that is the …, … band at the end of the gel
28S; large subunit; 18s; small subunit; mRNA; small RNA
Since RNA is susceptible to RNAses in cell and environment, it has to get isolated in the presence of a strong denaturant
- after isolation, we have all the RNAs that were present within the cell –> This doesn’t tell us if our sample is …/…, but if we don’t see … and …, that means the sample is pretty degraded
intact/degraded;
18S;
18S
mRNA formation:
DNA copied into primary transcript which is then spliced (… removed), … added along with … (which is a … monomer flipped in the opposite direction) to form mRNA
introns;
poly A tail;
5’ cap;
GTP monomer
one of the ways we can isolate mature mRNA is to utilize … which is typically just seen in mature mRNAs
- sometimes might accidentally get …. though
- can use beads that have … on them (…), enabling … to occur - use a column
poly A tail;
rRNAs;
dTs; multiple Ts;
hybridization
With isolated RNA:
can probe it to see if contains a particular transcript:
- …: run all RNA out on a gel to separate based on … and then transfer it to a membrane and then probe membrane with … and see where it was on the gel. By doing that, could identify how … expressed mRNA was for that sequence
northern assay;
size;
complementary sequence; big
with isolated RNA:
can probe it to see if it contains a particular transcript:
- … assay: probe mixed in with …, … then added such that everything else is degraded except where probe bound to complementary sequence
enables them to determine … of a particular sequence was in isolated sample
nuclease protection assay;
RNA;
RNAse;
how much
with isolated RNA:
qT-PCR:
…
able to calculate … of a transcript in a particular cell
quantitative reverse transcriptase;
number of copies
with isolated RNA: RT-PCR: - ... - this is what we're doing - 1st step is use of ... to make cDNA - 2. cDNA obtained that is more ... than original RNA, enables us to have expressed sequence without ...
reverse transcription pcr;
reverse transcriptase;
stable;
introns
with isolated RNA:
RT-PCR:
- 3. Use PCR, which uses …, … that work with polymerase to copy sequence they’re bound to
- gives us lots of copies fairly quickly (… - … hrs)
- … is template for PCR
DNA polymerase;
primers;
4 - 5;
cDNA;
with isolated RNA: RT-PCR: - primers made to only amplify ... - polymerase adds to ... end of primers - use of gel afterward doesn't provide ... information, just indicates ...
one specific sequence;
3’;
quantitative;
presence of sample
with isolated RNA:
in vitro translation:
- important if you have a protein … - if you were to try and express it, it would … cell because it regulates … or it produces something that …
toxic to the cell;
kill;
cell cycle;
jams up translation
with isolated RNA:
in vitro translation:
- also enables you to produce … of a particular protein if you wanted to do further studies
~
include … in gel - looking to see that one single band is formed and then we want to see the size of that band. it should be in the … range, which is the size of the expressed sequence
a lot;
size ladder;
1131
Because mRNA is smaller, it’s easier to amplify in the lab than …
isolating GFP plasmid:
- contains promoter for a virus that enables it to be transcribed in a … - viral promoter though, so can’t control the …
- MCS: … site - that is where … sequence will be inserted
genomic DNA; mammal; amount being transcribed; multi cloning; actin
Need to make sure that actin sequence is in the same … to ensure that we have fusion protein that has … and actin in it
reading frame;
GFP
plasmid has …, … resistance genes that enables us to select for cells that have this plasmid by growing them in the presence of … and …
kanamcyin;
neomycin;
kanamycin;
neomycin
Take 1 mL culture and put in eppendorf and centrifuge:
- tubes need to be balanced when spinning
- spinning pellets bacteria to bottom of tube
- when using cell resuspension: want to make sure there’s no … when doing this
- cell lysis solution is …: will get rid of … (denature) which prevents …, important for isolating … vs. …
pellet; basic; dsDNA; base pairing; plasmid; cell's genomic DNA
cell lysis solution is basic:
- plasmids will be able to … and can be …
- with cell lysis it should become more viscous - means that there’s a lot of … in the sample
base pair;
isolated;
DNA
after cell lysis solution, use … solution:
- active at …
- kills off … that might …
- if lysis step is over 5 mins, bases are … and DNA impacted
alkaline protease; high pH; proteins; nick DNA; changed
After alkaline protease solution, … solution added to bring …, enabling DNA to … with … sequence so that we have the … again
neutralization; pH back down; hybridize; complementary; ds plasmid
after adding neutralization solution, proteins and membranes begin … We therefore … to take those and cement them to side of tube
precipitating;
centrifuge
after centrifuging to precipitate out proteins and membranes, transfer cleared lysate to … which has a … membrane (…) at the bottom, interacts with … charges on DNA
- can’t transfer pellet bc then it will cause some DNA to be …
- incubate
spin column;
charged; +;
negative;
lost
after transferring cleared lysate to spin column, … spin column
then …
centrifuge;
wash
washing spin column:
column wash with … that keeps DNA precipitated on membrane so it stays on membrane and everything else is collected in …
after each wash, dump out what’s in collection tube
ethanol;
collection tube;
after washing, … - centrifuge for 2 mins
this is done so that there’s no … in final eluate. This is bc that compound can interfere with reactions down the road and also causes sample to jump out of gel
dry spin;
ethanol;
after dry wash, transfer spin column to new tube. add water, spin it down and then keep the tube
gel to ensure that it’s the right … and right … -> how plasmids move through gel is impacted by …
size;
banding pattern
how plasmids move through gel impacted by shape:
… form and … form
we want to see the bright bands that correspond to those forms
supercoiled;
nicked
