Plasmid Subcloning

Purpose:

The method is used to clone smaller portions of inserts (up to 15 kb in size)which previously have been cloned in YACs phage cosmids or other plasmids.

Time required:

Plasmid Vector Preparation

Restriction digest and Calf Intestinal Phosphatase (CIP)reaction - 4-6 hours or overnight

Gel electrophoresis- 4-8 hours or overnight

Fragment elution and purification - 4-6 hours

Insert Preparation

Restriction digest -4-6 hours or overnight

Gel electrophoresis- 4-8 hours or overnight

Fragment elution and purification - 4-6 hours

Ligation Reaction - minimum of 4 hours,best overnight

Special Reagents:

10X CIP Buffer

10X Ligation Buffer

Preface:

Bacterial plasmids are double-stranded circular DNA molecules of 1- 200 kb in size that replicate and are inherited independently of the bacterial chromosome.Different plasmids replicate to different extents in their host some reaching a copy number as high as 700 per cell.They function as accessory genetic units frequently containing genes coding for enzymes that are advantageous to the bacterial host.The most commonly conferred phenotype by plasmids is antibiotic resistance though plasmids can also code for antibiotic production restriction and modification enzymes colicins enterotoxins and enzymes used to degrade complex organic compounds.More than one plasmid can occur in a single cell but since they compete for the same set of replication enzymes one plasmid usually dominates the others.Over the course of a few generations the minority plasmids are completely eliminated and the descendants of the original cell will contain only one of the original plasmids.Over 30 different "incompatibility" plasmid groups have been identified.

Under natural conditions many plasmids are transmitted to a new host by a process known as bacterial conjugation.Newer plasmid vectors however lack the nic/bom site and cannot be conjugated.In the laboratory plasmid DNA can be introduced into modified bacteria (called competent cells)by the process of transformation.Even under the best circumstances plasmids become stably established in a small minority of the bacterial population.Transformants can be easily identified by the selectable marker encoded by the plasmid i.e.antibiotic resistance and the resulting phenotype of those bacterial cells.

Uncut plasmid DNA can be in any of five forms - nicked circular linear covalently closed supercoiled or circular single-stranded.When run on a gel one frequently will see these forms as different bands.The exact distances between the bands of these different forms is influenced by percentage of agarose in the gel time of electrophoresis degree of supercoiling and the size of the DNA .One cannot accurately determine size from uncut plasmid DNA .When cut with an enzyme with one recognition site in the plasmid almost all the DNA will fall in one band which equals the linear size of the plasmid (see illustration below).

Almost all research plasmid vectors contain a closely arranged series of synthetic restriction sites called the "polylinker".In most cases these restriction sites are unique to the polylinker and hence provide a variety of targets that can be used singly or in combination to clone foreign DNA fragments.The most recent phase of construction of plasmid vectors involves the incorporation of ancillary sequences that are used for a variety of purposes including generating single-stranded DNA templates for DNA sequencing transcription of foreign DNA sequences in vitro and expression of large amounts of foreign protein in vitro.

Fragments of foreign DNA can be cloned in a linearized plasmid vector bearing compatible ends by the activity of Bacteriophage T4 DNA Ligase.The enzyme will catalyze the formation of a phosphodiester bond between adjacent nucleotides if one nucleotide contains a 5'-phophate group and the other nucleotide contains a 3'-hydroxyl group.