L15 - Classical gene cloning 2 Flashcards
(17 cards)
why can we only make genomic libraries for prokarytotes
only has coding regions
= each cut fragamemt that goes into a different vector codes for a different protein
what are comnpatible sticky ends
different restriction enzymes that cut at different recognition sites but leave the same overhanging sequences
= even tho original recognised sequences arent the same
= the produced ligation of these comptabile sticky ends may produce a sequnc that can not be recongised by the orignal enzymes
why do we not want our inserted gene/DNA to be too small or too big BUT just right (goldiocks zone)
too small:
genes may not have a promoter or may be so small they are only ‘part’ of a gene
= hard to select for as may not code for something itself
too big:
multiple genes within it
= makes it hard to tell which gene is within a vector as there are multiple phenotypes
what is DNA shearing
physically breasking the DNA by forcing through narrow space
= fine-gauge needle for example
randomly rips the DNA at non-specific sites producing fragments of different sizes with blunt ends
what is a partial digest
treating DNA with less enzyme OR for less amount of time to prevent the DNA being ‘fully digested’:
restriction enzymes cuts at a specific sequence (GAATTC)
In a complete digest, all GAATTC sites are cut.
In a partial digest, only some GAATTC sites are cut
= results in mixture of fragment sizes some overlapping
compare partial digest to shearing
both methods give you a range of overlapping fragments of various sizes
Partial digest gives controlled randomness - you know what sites are being cut
Shearing gives true randomness - which can be helpful, but is less precise
= PD gives sticky while shearing creates blunt
name the 3 ways to prevent vector recirculating/religation
Alkalaine phosphatase:
removes 5’ phosphate from vector –> 3’ OH cannot bind to other end = insert retains 5’ phosphate so can bind
Double digest:
one end is cut with one RE and the other is cut with another RE = these ends are not compatabile
HOWEVER the insert is cut with the same 2 REs = the insert will have one end complimentary to one side and another end complimnetarty to the other side
lethal gene disruption:
vector contains a lethal gene in MCS = if religation occors the cell dies due to gene being intact
what is the advantage of uysing a double digest to prevent recircurisation/ religation of the vector
forces directionality
= the end of the plasmid cut with a specific RE has to bind to the end of the inset cut with the same RE
the other end is not complimentary due to incompatible sticky ends from different REs
what is transformation
changing the phenotype of an organism
= inserting a plasmid/peiece of DNA into a cell
what is the goal of using a vector mixture to transform bacteria
1 plasmid per bacteria due to the very low efficiency of transformation
= unlikely you’d insert 2 plasmids into 1 cell
means that each colony is from a bacteria with a single insert = this is Good as multiple plasmids would make it hard to know whichj insert is causing which phenotype
why do we not need to worry about multiple plasmids going into a simgle cell in transformation - 2 reasons
- transformation is a very inefficient proscess = your lucky to get 1 !
- plasmids can take over one another:
similar plasmids may fight for replicative machinery = one is likely to be more stable and dominant
2 forms of transformation
Electroportation –> temporaliy polarise membrane
chemical transformation –> heat shock
what is a phenotypic slection method used to check if a genomic library is good quality
XGAL
= blue/white selection to check the inserted DNA is present
done after screening for antibiotic resistance (example) to ensure plasmid is present
pros and cons for screening for phenotypic slection (XGAL and antibiotic restance)
+:
quick and straightforward
no prior info on the sequnce needed
-:
a strong selection method is required = antibiotic resitamce or disruption of gene (b-galactosidase)
what are the 2 methods for clone selection
Phenotypic selection
Western Screening
describe what is and how to do a western blot
form of clonal slection screening when you DONT have a strong phenotypic selection
- induce protein expression (IPTG for example)
- lyse cells and run proteins through SDS-PAGE
- transfer proteins from gel onto PVDF or nitrocellulose membrane
- incubate in blocking buffer (milk) to preveny non-specific antibody binding
- apply primary antibody to bind to desired/selcting for protein
- add antibody labbelled with enzyme that binds to 1st if present
- prescence is checked by adding substrate that enzyme breaks down
= emits light
8. viewed on X-ray film = if band shown = protein/gene is present in clones
pros and cons of western blotting as a clonal selection method
+:
do not need to know anything about sequnce and is very accurate
_:
expensive and time consuming
