Quiz 3 Info Flashcards
We did the Topo Ta cloning to give actin sequence … (??) so that we can then insert actin into the …
restriction sites (not sure if this is even correct); gfp plasmids
PCR on bacteria from colonies. Touching pipet to colony of plate and putting that in PCR
- cycles of heating and thawing will … and …, such that it can then be used as template for PCR
- If we were to do this, we would still have to …
break bacteria; release plasmid;
culture the right colony
EcoR1 and Sac2 restriction sites in … and … primers for actin, respectively
To design primers, had to see what restriction sites are in … but not in … so that actin coding sequence can be flanked by those restriction sites
forward; reverse;
MCS;
actin sequence
have to … gfp plasmid by cutting it with …
- cutting it with ecor1 and sac2 as well to generate …
linearize;
restriction enzymes;
same sticky ends
ecor1 and sac2 will always cut at the same restriction site so long as … aren’t wildly off
- for ecor1, cuts between … and …
- for sac2, cuts between last … and … when it sees its particular restriction site
salt concentrations;
g; a;
c; g
we ran out plasmids against 1 kb ladder - technically not the right control for plasmids bc 1 kb ladder is made of … fragments of DNA which will migrate differently than supercoiled/nicked DNA.
- nicked will move … and supercoiled will move …. in gel and will move as though it’s … than it really is
linear;
larger than apparent size;
faster;
smaller
if pcr 2.1 vector just seals back on itself, it will be … bp
- supercoiled will be migrating such that it’s just over the … band
- if they have actin sequence, supercoiled will be migrating almost as far as … bp band in the ladder
- actual size is …
3929;
2000 bp;
3054;
5071
pcr 2.1 vector:
better control would be to use … in one of the lanes and if our plasmid doesn’t migrate as far, it would indicate that we have …
blue colony; DNA
Did M13 to take a look at what might be … in the plasmids
- plasmid has m13 sequences on it that are about … bp apart - if no insert, will get product at …
- if insert is actin - we’re adding … bp such that the total size is … bp
inserted; 200; 200 bp; 1142; 1343
… that actin was inserted didn’t matter when making too plasmid
- now it matters when inserting into gap plasmid
- has to be in same … as gfp before it, as well
direction;
reading frame
this time, we’re cloning into a … plasmid - have to … first, because right now it can’t take up any DNA
closed;
cut into them
cloning into a closed plasmid:
… comes after gfp coding sequence, which is after … sequence
- we cut the plasmid by using … cutting into …
multi cloning site; GFP coding; promoter; restriction enzymes; restriction sites
use restriction enzymes to cut plasmid:
- cutting ends of actin so it has … Cut gfp plasmid with the … so it has compatible … to it
specific overhangs;
same restriction enzymes;
overhangs
Nucleases:
- break … in nucleic acid
- substrate depends on …
phosphodiester bonds;
enzyme itself
Nucleases:
- substrate depends on enzyme itself:
- … - break down RNA
- … - break down DNA
- … - only cut at the ends
- … - cut in the middle of a sequence
ribonuclease;
deoxyribonuclease;
exonuclease;
endonucleases
Nucleases:
- endonucleases: we’re using …, which binds to the particular sequence of nucleotides and cuts …
type 2;
inside of that sequence
Nucleases:
we’re using type 2 endonucleases:
- … - cut on …
- their restriction sites are … - if you read them … on either strand, they will have same sequence
dimers;
both strands;
palindromes;
5’ to 3’
restriction endonucleases evolved to protect bacteria from … so that if there was any linear DNA that entered, the endonucleases that were endogenous to the bacteria could attack and … that was infecting the cell
- bacteria protects its own DNA by … enzymes that recognize the same sequences as restriction enzymes, but add … on DNA so that restriction enzymes don’t cut it
viruses;
break down that DNA;
modification;
methyl groups;
restriction site = …
- for type 2, they are the same because enzymes bind and cut …
- for the ones we’re using, they are … nucleotides long
- … = palindrome
recognition site;
within the same sequence;
6;
inverted repeat
binding and cutting is very specific as long as … are right
- how often DNA will be cut depends on how often … is in DNA –> probability is higher for … restriction site sequences
salt cones;
restriction site sequence;
shorter
use restriction enzymes for:
- …
- …
restriction mapping;
cloning
Use restriction enzymes for:
- restriction mapping: comparing two pools of DNA by cutting them with … –> if the DNA is the same, will get … Otherwise, will get …
the same enzymes;
same fragments;
different fragment lengths
we’re cutting gfp and actin pcr product so that we can …
ligate them together
using … primers that also have …
- we’re adding … onto forward and reverse primer
gene specific;
restriction site sequence;
restriction site;
using gene specific primers that also have restriction site sequence:
we’re adding restriction sites onto forward and reverse primer:
- … restriction on forward
- … restriction on reverse
^ this is to ensure that actin will …
upstream;
downstream;
go in the right direction
using gene specific primers that also have restriction site sequence:
we’re adding restriction sites onto forward and reverse primer:
- have an additional nucleotide to ensure it’s in the …
right reading frame
