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Biotech > Quiz 3 Info 2 > Flashcards

Quiz 3 Info 2 Flashcards

1
Q

The easiest way to see if there’s been digestion is to look at what’s happening with the …

  • undigested: see … and … bands
  • digested: … migrating further than … but not as far as …
A 
plasmid; 
supercoiled; nicked; 
linear band; 
nicked; 
supercoiled
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2
Q

if plasmid is only being cut by one of the restriction enzymes, that means it has the …

  • when we see extra bands, we have to redo the digests
  • we’ll be plating onto lb agar plates with … such that only bacteria with the gfp plasmid survive
A

same sticky ends;

kanamycin;

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3
Q

we’ll be plating onto lb agar plates with kanamycin such that only bacteria with gfp plasmid survive
- but PCR 2.1 plasmid that we cloned into can also survive so if there’s … with the actin product, that can give us … too

A

contamination;

colonies

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4
Q

if multiple bands for plasmid, reediest with

A

both enzymes

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5
Q

we calculate concentrations using …

A

low mass ladder

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6
Q

Bc sac ii isn’t completely cutting we have:

  • actin: some that have … and some that only have …
  • plasmid: some plasmids with…, some plasmid …
A

both sticky ends;
eco r1 sticky end;
both sticky ends;
without sac ii sticky ends

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7
Q

sac ii expires faster - expiration date is accurate to estimating …

A

decreased activity

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8
Q

how to design primers:

  • find … from ncbi
  • find … –> shows where … and … is when you click on cds, this is you just want to copy coding sequence
A

gene sequence;
coding sequence;
ate;
stop codon

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9
Q

how to design primers:

  • there is a link to protein sequence - tells you how many aa there are
  • number on the left side in sequence tells you the position of the first nucleotide.
  • for designing primers, we want one that’s on or before … for forward and a reverse primer that’s on or after …
A

start tag;

stop codon

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10
Q

how to design primers:
- 20 nucleotides at the start and 20 at the end and then check if … and … are balanced and then move things around to get the right

A

gs; cs;

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11
Q

how to design primers:

  • take sequence (including primers I think?) and put it into nebcutter. Find restriction enzymes that don’t cut into …
  • compare that list to what’s in the …
  • left with enzymes that we can …
A

sequence we need;
multi cloning site;
add to primers

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12
Q

to choose which to add to primers:

  • … on forward
  • … on MCS should go on reverse
  • this ensures that pcr product goes in the right direction
A

upstream;

downstream

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13
Q

to choose which to add to primers:

  • if you have primer that starts on tag, try to find enzymes that fall right into … so you don’t have to …
  • restriction sites for 5’ - consider reading frame of gene sequence … (want to keep tag together) and reading frame of …
A

reading frame;
add nucleotides;
within primer;
MCS

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14
Q

if you have a list of restriction sites that you can add, put upstream restriction site on … and downstream restriction site on … to ensure that everything goes in …

A

forward primer;
reverse primer;
right direction

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15
Q

when cloning into expression vector, understand how mcs works. understand that things need to be in the … to get the fusion protein.
- gene that we’re cloning in needs to be going into the plasmid in the right …

A

same direction;

direction

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16
Q

understand what restriction enzymes are and the restriction enzyme we’re using are type …

  • they are …, bind to specific site and cut … that site
  • cutting …
A

2;
dimers;
within;
phosphodiester backbone

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17
Q

understand what restriction enzymes are and the restriction enzymes we’re using are type 2:

  • binding and cutting is specific to the restriction enzyme in that it should only bind and cut …
  • even having difference of just one nucleotide will change … of that restriction site and restriction enzyme should no longer be able to …
A

within that particular sequence;
the shape;
bind and cut

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18
Q

understand what restriction enzymes are and the restriction enzymes we’re using are type 2:
- problems can cause …

A

star activity

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19
Q 
understand what restriction enzymes are and the restriction enzymes we're using are type 2: 
star activity can be caused by: 
- too much ... 
- too much ...
- ... concentrations are off
- ... in the reaction
A

enzyme;
glycerol;
buffer;
ethanol

20
Q

understand what restriction enzymes are and the restriction enzymes we’re using are type 2:
- minimized star activity by … and ensuring it was in … and not in another rxn so that we didn’t have to worry about making concentrations right and modifying them bit by bit. Tried be careful with … to ensure that we weren’t adding too much enzyme or glycerol. Also did extra … to get rid of any residual … off the column prior to eluting the DNA off the column

A 
purifying DNA; 
water; 
pipetting enzyms; 
dry spins; 
ethanol
21
Q

first ligation we did was topo ta

  • topoisomerase was bound, there were t overhangs
  • for this we didn’t care about the direction that the pcr product went into the plasmid bc it wasn’t an … –> we were only interested in taking the expressed sequence and putting it into a plasmid where we’d be able to make a lot of copies of it …
A

expression vector;

quickly

22
Q

first ligation we did was topo ta

  • we’re considering how much pcr product we were adding to the cloning rxn
  • we had much more of our … than of the … - trying to …
A

pcr product;
plasmid;
push pcr product into the plasmid

23
Q

this time, we need to be able to create …. on our pcr product so that it can ligate with the vector that we’re cloning into
- last time, we coded into a plasmid that was already … This time, we didn’t

A

overhangs;

open

24
Q

We had to make a piece of DNA with gene sequence and figure out a way to put it into the GFP expression vector in the right direction and reading frame.
- we have to cut plasmid open and cut pcr product - this was the …

A

digestion

25
Q

now we have the two different … on each side to actin pcr product and gfp plasmid such that those two can only ligate together …

  • based on …, we’ve engineered this so that it’s in the right reading frame
  • *** review/practice primer design **
A

sticky ends;
in one orientation;
primer design

26
Q

ligase that we’re using this time is …

- enzyme isolated from a … - makes a … bond between … and …

A

T4 ligase;
bacteriophage;
phosphodiester;
3’ OH; 5’ phosphate

27
Q

ligase that we’re using is T4 ligase:
makes a phosphodiester bond between 3’ OH and 5’ phosphate
- ligase has … with … group at the end

A

lysine;

amino

28
Q

how T4 ligase works:

- Break down … and add … onto … group of ligase

A

ATP;
AMP;
amine

29
Q

how T4 ligase works:
- Break down ATP and add AMP onto amine group of ligase such that … from phosphate attacks and … - wakes up … which then …

A

oxygen;
AMP transfers onto phosphate;
3’ OH;
attacks phosphate

30
Q

how T4 ligase works:
- break down ATP and add AMP onto amine group of ligase such that oxygen from phosphate attacks and AMP transfers onto phosphate - wakes up 3’ OH which then attacks phosphate –> … and … is formed

A

AMP gets released;

phosphodiester bond;

31
Q

how T4 ligase works:

  • ATP that we need for this rxn is already in the …
  • we need a happy medium of temp - ligase is most effective at …
  • if we used something that made …, the ideal temp for ligase would be fine
A

buffer;
room temp (25 deg. C);
blunt ends

32
Q

how T4 ligase works:

  • we need a happy medium of temp - ligase is most effective at room temp
  • if we used something with blunt ends, 25 deg C would be fine. But here, ideal for sticky ends is around …-… deg. C –> if both ends were blunted, we couldn’t control …
  • sticky (aka … ends) ends make it easy to control for …. that DNA is inserted into the plasmid
A

12; 16;
direction;
cohesive;
direction

33
Q

how T4 ligase works:

  • need a happy medium for temp
  • we achieve that by ligating at … deg C for 5 mins, and after that, we take a sample from ligation and add it to … and … the cells
  • won’t throw out ligation rxn - store them in the fridge
A

25;
competent cells;
transform;

34
Q

ligase buffer ensures that … and … is fine

- … as much insert as vector

A

buffer;
salt conc;
3x

35
Q

ligase buffer ensures that buffer and salt conc is fine:

  • 3x as much insert as vector
  • total DNA will be up to … in the rxn
  • … of ligase is excessive
A

100 ng;

1 microL;

36
Q
  • dealing with … bc we want 3x as many … of actin as plasmid
  • … ratio is only for sticky ends. If it was blunt ends, we’d … the ratio
A

fmol;
ends;
3:1;
increase (up to something like 5:1 or 10:1)

37
Q

do fmol calc for both insert and vector:

- fmol ratio depends on …–> might need to use a smaller ratio if you …

A

how much DNA you isolated;

purified very little DNA

38
Q

do fmol calc for both insert and vector:
- what’s limiting how much DNA we can add is that we’re keeping the rxn at … –> leaves … that we can fill with insert + plasmid and then water

A

20 microL total volume;

17 microL

39
Q

low mass ladder also indicates …

- match … of our product in the gel to that of the bands in LML

A

concentration;

brightness;

40
Q

low mass ladder also indicates conc:
- match brightness of product in gel to brightness of bands in LML –> divide standard conc from LML by … (since we loaded …)

A

3 microL;

3 microL of DNA into the gel sample

41
Q

low mass ladder indicates conc:

  • match brightness of product to brightness in LML and divide by 3 microL
  • divide ng amount from fmol ratios by … calculated based on …
A

ng/microL;

LML

42
Q

Controls:

- to test if we got ligation: use …. If there are colonies, … If there are no colonies …

A

pEGFP-EcoRI (probably means just digest it with one thing to check if ligase works);
ligase works;
no ligase

43
Q

Controls:
- to test if the cells are competent, use …
If there are colonies: …
No colonies: …

A

pEGFP;
yes, competent;
bad cells

44
Q

Controls:

- to try and distinguish if there’s insert: … and … controls

A

plasmid alone;

actin alone

45
Q

Controls:
- plasmid alone control:
if colonies on plasmid alone control, that means it was …
- if cut two times, will get a … piece of DNA —> … bc it’s not …

A

only cuts ne time;
linear;
no colonies;
circular

46
Q

Controls:
- actin alone:
should definitely not get … because there’s no … -> shouldn’t be … and shouldn’t be able to … cells and …

A

any type of colony;
plasmid info;
able to transform cells;
produce colonies

47
Q

Controls:
- actin alone: if there are colonies here, … is still around. If no colonies, that means that there’s … in there, as expected

A

template DNA;

no additional plasmid

Biotech (17 decks)

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