Quiz 3 Info 2 Flashcards
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 …
plasmid; supercoiled; nicked; linear band; nicked; supercoiled
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
same sticky ends;
kanamycin;
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
contamination;
colonies
if multiple bands for plasmid, reediest with
both enzymes
we calculate concentrations using …
low mass ladder
Bc sac ii isn’t completely cutting we have:
- actin: some that have … and some that only have …
- plasmid: some plasmids with…, some plasmid …
both sticky ends;
eco r1 sticky end;
both sticky ends;
without sac ii sticky ends
sac ii expires faster - expiration date is accurate to estimating …
decreased activity
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
gene sequence;
coding sequence;
ate;
stop codon
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 …
start tag;
stop codon
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
gs; cs;
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 …
sequence we need;
multi cloning site;
add to primers
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
upstream;
downstream
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 …
reading frame;
add nucleotides;
within primer;
MCS
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 …
forward primer;
reverse primer;
right direction
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 …
same direction;
direction
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 …
2;
dimers;
within;
phosphodiester backbone
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 …
within that particular sequence;
the shape;
bind and cut
understand what restriction enzymes are and the restriction enzymes we’re using are type 2:
- problems can cause …
star activity
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
enzyme;
glycerol;
buffer;
ethanol
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
purifying DNA; water; pipetting enzyms; dry spins; ethanol
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 …
expression vector;
quickly
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 …
pcr product;
plasmid;
push pcr product into the plasmid
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
overhangs;
open
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 …
digestion
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 **
sticky ends;
in one orientation;
primer design
ligase that we’re using this time is …
- enzyme isolated from a … - makes a … bond between … and …
T4 ligase;
bacteriophage;
phosphodiester;
3’ OH; 5’ phosphate
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
lysine;
amino
how T4 ligase works:
- Break down … and add … onto … group of ligase
ATP;
AMP;
amine
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 …
oxygen;
AMP transfers onto phosphate;
3’ OH;
attacks phosphate
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
AMP gets released;
phosphodiester bond;
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
buffer;
room temp (25 deg. C);
blunt ends
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
12; 16;
direction;
cohesive;
direction
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
25;
competent cells;
transform;
ligase buffer ensures that … and … is fine
- … as much insert as vector
buffer;
salt conc;
3x
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
100 ng;
1 microL;
- 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
fmol;
ends;
3:1;
increase (up to something like 5:1 or 10:1)
do fmol calc for both insert and vector:
- fmol ratio depends on …–> might need to use a smaller ratio if you …
how much DNA you isolated;
purified very little DNA
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
20 microL total volume;
17 microL
low mass ladder also indicates …
- match … of our product in the gel to that of the bands in LML
concentration;
brightness;
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 …)
3 microL;
3 microL of DNA into the gel sample
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 …
ng/microL;
LML
Controls:
- to test if we got ligation: use …. If there are colonies, … If there are no colonies …
pEGFP-EcoRI (probably means just digest it with one thing to check if ligase works);
ligase works;
no ligase
Controls:
- to test if the cells are competent, use …
If there are colonies: …
No colonies: …
pEGFP;
yes, competent;
bad cells
Controls:
- to try and distinguish if there’s insert: … and … controls
plasmid alone;
actin alone
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 …
only cuts ne time;
linear;
no colonies;
circular
Controls:
- actin alone:
should definitely not get … because there’s no … -> shouldn’t be … and shouldn’t be able to … cells and …
any type of colony;
plasmid info;
able to transform cells;
produce colonies
Controls:
- actin alone: if there are colonies here, … is still around. If no colonies, that means that there’s … in there, as expected
template DNA;
no additional plasmid
