Cloning vectors

pUC vector

A plasmid vector. Contains MCS in Lax Z alpha. BW selection after induction with IPTG and X-Gal metabolite.  Ampicillin resistance.  Only compatible with certain E.coli strains.

DNA extraction 

Alkaline lysis from cells.  Resuspend pellet. Lyse cells - lysozyme to break open cells, SDS to complex with protein, NaOH to ss DNA.  Neutralise lysate with buffer - precipitates cellular components, leaving plasmid DNA in solution. Bind DNA from supernatant to matrix (silica surface) in column by spinning.  Wash matrix to remove non DNA products. DNA is removed from silica surface by water. 

Expression vectors

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Direct transcription and translation of a cloned gene. Promoter included. A tag and cleavage site are included to aid isolating the desired protein in a column, tag can consequently be cleaved off. This is located between MCS and promoter. Strong or weak promoters can be used to regulate the level of expression. E.g. Ptac, induced by IPTG.GFP (green fluorescent protein - UV light) is also a selectable marker

More on plasmid vectors

We can clone a promoter in front of a reporter gene e.g. Lac Z, in order to test conditions in which promoter is working. A shuttle vector can be used to move a clone between cell types, contains both mammalian and E.coli Ori, along with a mammalian virus origin. Gateway cloning: Commercial setup, introduce DNA into entry clone. From the entry clone, may be recombined into almost any sort of cloning system e.g. mammalian or viral or yeast ect... no restriction enzymes or ligase...

Cloning PCR products & Gibson assy

Restriction cut sites added to the ends of primers OR, TA cloning: tac polymerase adds A onto 3' ends of PCR product, and a vector with 3' T overhang (cant re-lygate).The gibson assembly is a commercial system that assemble multiple DNA fragments in one reaction. Allowing for insertion of multiple fragments into a vector as one.  

Cosmid vector

A plasmid vector, with a lambda cos site.  Ori MCS Antibiotic resistance  cos site for lambda DNA packaging  can package up to 50Kb Method of cloning - linearise cosmid at restriction site and add DNA. Ligate all cosmids together. In vivo phage packaging recognises cos sites 35-45Kb apart. Infect E.coli and re-circularise. The phage shell is made in bacteria using 2 strains: one with mutations to allow only head and tail production respectively. Protein extracts from the 2 cultures are taken and added to cosmid DNA. These have the advantage of efficient introduction to bacteria (host).  

BACs: Bacterial artificial chromosomes

Really big fragments.  Low copy number.  Containing stabilizing genes.  Antibiotic resistance marker.  75-300Kb inserts.  based on the F plasmid, it contains parA,B and C proteins to stabilize it. May include colour selection (Lac Z a). These were developed for genomic cloning.