iGEM 101: Session 2

2/19/15 Jarrod Shilts2/22/15 Ophir Ospovat

2. Miniprep

▪ Extraction and purification of plasmid DNA from bacteria

▪ Different sizes depending on desired yield– Mini, Midi, Maxi, Mega, Giga– Protocols vary minimally

▪ Several different methods– Column (spin, vacuum)– Phenol chloroform– Cesium chloride gradient– Salt/alcohol precipitation

Relative Time vs. Purity

Universal Stages

▪ Growth of Bacterial Culture

▪ Harvesting and Lysis

▪ Purification of plasmid DNA

Bacterial Growth

▪ Incubation at 37 ° C for 12-16 hours at 200 rpm

▪ Bacteria should be in stationary phase

▪ Culture 1-5 mL

Alkaline Lysis Solution I (GTE)

▪ Glucose maintains osmotic pressure

▪ Tris buffers cell

▪ EDTA weakens membrane

▪ RNase

*EDTA also chelates Mg2+ needed by nucleases

Resuspension

Alkaline Lysis Solution II (SDS/NaOH)

▪ NaOH lyses the cells

▪ SDS dissolves lipids and proteins

▪ Under alkaline conditions, DNA denatures

Lysis

Alkaline Lysis Solution III (KAc/AcA)

▪ Acetic acid neutralizes solution– Renatures plasmid DNA selectively

▪ Potassium acetate precipitates SDS– Traps chromosomal tangle

▪ Solution contains plasmid, small contaminants, and chromosomal fragments

Precipitate

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 remaining SDS

▪ 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+

▪ Store at 4 oC or colder

Elution