igem 101: session 4 3/19/15jarrod shilts 3/22/15ophir ospovat
TRANSCRIPT
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iGEM 101: Session 4
3/19/15 Jarrod Shilts3/22/15 Ophir Ospovat
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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
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Gel Extraction Specific
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Universal Steps
▪ Resuspension in suitable liquid environment
▪ Binding to Column
▪ Washing and Eluting
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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
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Chaotropic Solution
▪ Guanidium salt
▪ Tris buffer
Resuspension
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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
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Ethanol
▪ Removes salts and environmental contaminants
▪ Usually two washes performed
Washes
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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