Quiz 3 PPT Flashcards
directional PCR: PCR using primes that have … on them, making a new insert that will clone into another …, in the right … and the right …
restriction sites;
plasmid;
direction;
reading frame
directional pcr:
cloning into … plasmid
pEGFP
This time, we’re cloning into an … which is why the direction and reading frame become relevant
expression vector
how do we control direction and reading frame and open the plasmid?
… and their …
restriction enzymes;
restriction sites
nucleases: enzymes that …
degrade nucleic acids
nucleases - types: ... ... ... ...
ribonucleases;
deoxyribonucleases;
exonucleases;
endonucleases
restriction endonucleases:
- from …
- cut …
bacteria;
ds DNA
Restriction endonucleases:
- bind at … - the … - and cut it
- … bind the same sequence and … it
specific nucleotide sequences;
restriction site;
modification enzymes;
methylate
Restriction endonucleases:
- prevent invasion by … - sees particular sequence, cuts it. “restricts” … by …
- cuts at sites in DNA, not just nibble away at ends
foreign DNA;
infection;
viruses
Recognition sites
- same as …
- most are …, …, or … nucleotides long
- …
restriction sites;
4; 6; 8;
inverted repeats
… vs … restriction enzymes
type i; type ii
Frequency of cutting:
- depends on number of … in recognition site
- shorter sequences have a … than longer sequences, based on probability
- ** look at example in ppt ***
bases;
greater frequency
Uses for restriction endonucleases:
- …
- …
restriction mapping;
cloning
uses for restriction endonucleases:
- restriction mapping: … (…)
- cloning: …
restriction fragment length polymorphisms; RFLPs;
molecular scissors
what needs to be cut?
- … - ….
- …. -…
vector; pEGFP-C1;
PCR product; actin
the fragment with the right sticky ends comes from … - PCR with … + …
directional PCR;
gene-specific part;
restriction sites
how to choose restriction sites?
- what RS are … to be copied?
- what RS are in the …?
not already in sequence;
MCS of plasmid
superfi for directional pcr:
- …
- …, needs …
- different reaction conditions than GoTaq
proofreading;
inactive;
hot start
superfi for directional pcr:
- … is bound to SuperFi, need “hot start” (.. deg. C for 2 mins) to …
- this hot start should help with …, …, and …
antibody; 94; remove ab; specificity; sensitivity; yield
digestion reaction conditions:
- …, up to 1 microgram (… ng)
- …
- …
- …
DNA; 1000; buffer (10 x); water; enzyme
digestion reaction conditions:
- digest for … - … hrs, at … degrees C (up to … hours without … activity)
1; 3;
37;
16;
star
star activity?
- enzyme cuts at sequences …
other than its restriction site
star activity causes:
- excess …
- excess … (> …)
enzyme;
glycerol;
5%
star activity causes:
- high … to … of DNA (> …)
U (wth); micrograms; 100 U/microgram
