Quiz 1 Info Contd 2 Flashcards
(44 cards)
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
