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
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