Molecular Cloning Flashcards
(24 cards)
what is the purpose of gene cloning?
to isolate large quantities of a specific gene and their gene products
what are the steps in gene cloning?
- isolation and fragmentation of DNA
- joining desired DNA fragment to cloning vector
- introducing into host organism (transformation)
how are DNA fragments isolated?
PCR amplification or restriction
specific primers
what is different about DNA isolation primers?
at 5’ end they contain a specific restriction motif
what would happen if the fragment to be cloned contained internal restriction sites targeted by restriction enzyme?
fragment would be cut in half
when is PCR not required for DNA isolation?
if the donor DNA target gene is flanked by the desired restriction sites
how is self ligation in vector insertion avoided?
the 5’ end of the vector is dephosphorylated
what enzyme catalyses the ligation of a vector insert?
DNA ligase
what is the multiple cloning site?
region in the vector that is densely enriched of restriction sites
what are competent cells?
bacterial cells able to take up DNA from the environment with ease
bacillus and strep
how are cell prepared to be competent?
calcium chloride
electroporation (increase cell permeability)
what are vectors?
DNA molecules that transfer genetic material into a living cell so that it can be incorporated into the cell genome and expressed
what are vectors composed of?
origin of replication, multiple cloning site, selection marker
in what ways can vector choice impact cloning?
absence of:
- ORI
- RE recognition site
- selectable genetic marker
- gene for screening
what could improper DNA fragment preparation result in?
- incomplete RE digestion
- digested DNA as smear
- extra bands on gel
- no PCR amplification
what antibiotic selection markers does pBR322 contain?
ampicillin and tetracycline
what selection does pUC8 contain?
antibiotic resistance and inactivation of b galactosidase LacZ
screening of pUC8 clones shows what
resistance to ampicillin but unable to make b galactosidase
how is the b galactosidase assay performed?
X gal is broken down by b galactosidase to form blue
if b galac is inactivated white colonies will form
what are knockout mutations?
when a cassette is inserted into a gene, the gene loses its function and gain the function of the insert
what is a transcriptional unit?
a dna sequence encoding for a single RNA molecule
what does a transcriptional unit contain?
promotor
coding region
terminator
what is polycistronic mRNA?
single transcript encoding for multiple genes
preceded by 5’ UTR
prokaryotic transcription
RNA polymerase without primer
