1. iGEM 101: Session 2 2/19/15Jarrod Shilts 2/22/15Ophir Ospovat

2. 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

3. Relative Time vs. Purity

4. Universal Stages ▪ Growth of Bacterial Culture ▪ Harvesting and Lysis ▪ Purification of plasmid DNA

5. Bacterial Growth ▪ Incubation at 37 ° C for 12-16 hours at 200 rpm ▪ Bacteria should be in stationary phase ▪ Culture 1-5 mL

6. Alkaline Lysis Solution I (GTE) ▪ Glucose maintains osmotic pressure ▪ Tris buffers cell ▪ EDTA weakens membrane ▪ RNase *EDTA also chelates Mg 2+ needed by nucleases

7. Alkaline Lysis Solution II (SDS/NaOH) ▪ NaOH lyses the cells ▪ SDS dissolves lipids and proteins ▪ Under alkaline conditions, DNA denatures

8. 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

9. 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

10. Ethanol ▪ Removes salts and remaining SDS ▪ Usually two washes performed

11. Elution ▪ Usually done with water (60 °C) ▪ TE buffer also used when there are no downstream applications – Tris buffers the solution – EDTA chelates for Mg 2+ ▪ Store at 4 o C or colder