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25 Cards in this Set

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Genes are joined w/ a cloning vector so it can be transferred into a host cell, what are the 2 cloning vectors?
1)plasmid
2)bacteriophage
What are plasmids? (4 characteristics)
1)extrachromosomal
2)self-replicating
3)double stranded
4)circular DNA
Plasmid Cloning Vector pBR322 contains....
1)4,361bp's
2)ampicillin resistance gene
3)tetracycline resistance gene
pBR322 has recognition sites for ____(3) within the tetracycline resistant gene
HindIII
Sal
BamHI
pBR322 has recognition sites for ____(1) within the ampicillin resistant gene
PstI
pBR322 also has a ___ recognition site not within coding DNA
EcoRI
What is "insertional deactivation" of the pBR322 tetracycline gene?
putting foreign DNA into the TCN gene using a restriction site to inactivate TCN resistance
Regarding pBR322, the host cell containing the plasmid-cloned DNA construct is...
sensitive to TCN but resistant to ampicillin
Host cells that do not incorporate the plasmid cloned DNA construct...
don't survive in TCN or ampicillin
Host cell that incorporate recircularized(unmodified) pBR322 will be..
resistant to both TCN and ampicillin
Steps to ID those cells that contain plasmid-cloned DNA constructs (4) and 1 note
1)cells from transformation mix are put onto medium that contains ampicillin
2)only cells w/ pBR322 or pBR322 rDNA will live
3)the surviving cells are then transferred to a TCN plate
4)only cells w/ the recircularized(unmodified) pBR322 live
5)you have not just killed all your cells. the cells on this last plate were taken from a source plate. so if you have a plate w/ TCN, you have ID'd the colonies that have the unmod pBR322 on your source plate.
pUC19 has...(4)
1)lacZ'- gene of lactose operon of E. coli that encodes for B-Galactosidase
2)lacI- gene that produces repressor protein that regulates lacZ'
3)ampicillin resistance gene
4)14 different restriction sites
IPTG fxn
induces lac operon, making it so that lacZ' is not repressed by lacI and B-galactosidase is produced
pUC19 that is modified does what?
it is modified in the lacZ' region so that these cells can no longer produce B-galactosidase
Plasmid pUC19 selection procedure (5)
1)Introduce IPTG to cells so that the cells w/ recircularized pUC19 produce B-galactosidase
2)Introduce X-Gal to the cells
3)cells with unmodified pUC19 will turn blue due to rxn b/w X-Gal and B-Galactosidase
4)cells w/ modified pUC19 will be white
5)white colonies are the recombinant plasmids and thus selection is possible
Can human genes be put directly into bacteria?
NO, b/c we have introns and the bacteria can splice them out
cDNA properties (2)
1)No introns
2)produced by reverse transcriptase
A functional eukaryotic mRNA properties... (2)
1)no introns
2)poly A tail @ 3' end
How can small fraction of RNA that is mRNA be isolated from a mammalian tissue? (5)
1)RNA pass thru column w/ cellulose beads
2)short chains of thymidine bound to beads
3)poly A tails of mRNA bind to thymidine's
4)tRNA and rRNA pass thru
5)mRNA is unbound from beads using buffer w/ high salt []
To convert mRNA to double stranded DNA you need 2 enzymes
1)reverse transcriptase
2)DNA Pol I
RNA to cDNA steps (6)
1)reverse transcriptase uses oligoT primer and mRNA strand as template to make cDNA
2)this produces a DNA-RNA hybrid
3)RNA strand in the hybrid is removed by alkali or RNAase H
4)2nd DNA strand is made by DNA Pol I, starting @ the end of the hairpin loop
5)S1 nuclease then opens hairpin and degrades it
6)double stranded cDNA is formed
3 methods of IDing cells w/ target sequence
1)DNA hybridization using labeled DNA probe
2)immunological screening for protein product (using Ig's to the protein)
3)Screening for the protein activity (if product is an enzyme)
Screening for the protein activity mechanism
Add substrate for enzyme that produces a color product so you can SEE which colony has the enzyme
DNA hybridization screening steps (4)
1)From each colony on the original plate a sample is transferred to a nylon/nitro matrix
2)Cells on matrix are lysed, DNA is denatured, deproteinized and bound to matrix
3)labeled degenerate DNA probe is added to matrix
4)matrix processed by autoradiography to tell which cells have bound the labeled DNA
Immunologic screening method steps (5)
1)From each colony on the original plate a sample is transferred to a nylon/nitro matrix
2)cells on matrix are lysed and theirproteins are bound to the matrix
3)matrix is treated w/ primary Ig
4)unbound primary Ig is washed away, and secondary Ig (that has an enzyme attached to it) attaches to primary Ig
5)substrate for enzyme is added that produces a color enzyme, so you can SEE which colonies have the protein of interest