e coli cloning protocol

steps of protocol

amplify, digestion, miniprep, digestion, ligation, transformation

steps of digestion 1

incubate, purify insert, measure DNA

purpose of digestion 1

cut out gene from certain DNA and prepare the ends to fit into the plasmid

purpose of incubating under digestion 1

incubate insert w restriction enzymes at certain temperatures so DNA can insert properly

purpose of purification as step 2 of digestion 1

purify insert using PCR purification kit

purpose of measuring DNA as step 3 of digestion 1

quantify DNA

purpose of miniprep

purify vector from bacteria culture

purpose of digestion 2

in 1% agarose gel to quantify, verify, and confirm integrity of plasmid

purpose of ligation

join insert DNA and plasmid together, creating recombinant plasmid

purpose of transformation

incubating cells and ligation mixture in ice and heat shocks. Resuspend and plate cells

vector definition

DNA vehicle used to carry gene into the cell

plasmid definition

circular, original piece of DNA in the bacteria which you are going to insert the gene into

insert definition

gene you want to put into the DNA