iGEM 101: Session 4

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

2. 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

3. Gel Extraction Specific

4. Universal Steps • Resuspension in suitable liquid environment • Binding to Column • Washing and Eluting

5. 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

6. Chaotropic Solution • Guanidiumsalt • Trisbuffer Resuspension

7. 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 Chromatography

8. Ethanol • Removes salts and environmental contaminants • Usually two washes performed Washes

9. 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