igem 101: session 4 3/19/15jarrod shilts 3/22/15ophir ospovat

iGEM 101: Session 4

3/19/15 Jarrod Shilts3/22/15 Ophir 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

Resuspension

Transfer Solution to Column

Chromatography

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

Washes

Elution

▪ Usually done with water (60 °C)

▪ TE buffer also used when there are no downstream applications– Tris buffers the solution– EDTA chelates for Mg2+

Elution