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
