iGEM 101: Session 4 3/19/15Jarrod Shilts 3/22/15Ophir Ospovat

2. DNA Purification ▪ Purification of DNA from gel or enzymatic reaction ▪ Usually single size due to the nature of gels and PCR reactions ▪ Several different methods – Column (spin, vacuum) – Phenol chloroform – Cesium chloride gradient – Salt/alcohol precipitation

Gel Extraction Specific

Universal Steps ▪ Resuspension in suitable liquid environment ▪ Binding to Column ▪ Washing and Eluting

Gel vs. PCR Purification Specificities Gel -UV over-exposure -Careful cutting of DNA and gel -Solubilizing Buffer -Heating and cooling of gel PCR -Resuspension buffer first -Two elution volumes

Chaotropic Solution ▪ Guanidium salt ▪ Tris buffer

Transfer Solution to Column Silica -High salt conditions allow for the formation of a salt bridge of positive ions -Gel and DNA both negatively charged -DNA can be washed while bound Anion-Exchange -Relies on electrostatic interactions between positive beads and negative DNA -Eluted with high salt solution, not water

Ethanol ▪ Removes salts and environmental contaminants ▪ Usually two washes performed

Elution ▪ Usually done with water (60 °C) ▪ TE buffer also used when there are no downstream applications – Tris buffers the solution – EDTA chelates for Mg 2+