Plasmid and transformation

DNA cloning

process of making multiple copies of a specific fragment of DNA

• Gene of interest is inserted into a circular pice of DNA called a plasmid

Plasmid

Small, circular strands of DNA

4 parts of plasmid

1. Origin of replication

2. Resistance cassette 

3. Promoter region

4. Multiple cloning site

Origin of replication

DNA sequence that allows the plasmid to be replicated

Resistance cassette

provides a selection marker

• resistance to antibiotics(helps identify cells that successfully transform)

Promoter region

• signals start of transcription

• allows for expression of our gene of interest

• Often proceeded by multiple cloning sites

Multiple cloming site

gives a place to open a plasmid for insertion

Steps of DNA cloning

1. Isolation of target DNA fragments, get “inserts”

2. Ligation of inserts into a suitable cloning vector, inserts combine with plasmid

3. Transformation of recombinant plasmids into host organism, recombinant plasmids are introduced to host organism to multiple

4. Screening and selection of hosts with the desired recombinant plasmids into host, select host organisms that have successful recombinant plasmids

how to insert DNA into plasmid

• restriction enzymes: scissors, cuts DNA 

• DNA ligase: glue, joins ends

2 main types of transformation in lab:

electroporation: makes cell membranes permeable to DNA

Calcium chloride / heat-shock: chemically competent cells uptake DNA after heat shock

pGLO bacterial transformation

genetic marker - GRP gene

Resistance cassette - amp(ampicillin)

Arabinose - only grows when araC is prese/nt