MINIPREP

WHAT IS A MINIPREP? A plasmid DNA extraction from bacteria used to purify plasmid DNA-isolates plasmid in a highly purified form. Miniprep is used when the starting E. coli culture volume is 1~5 ml of LB broth and the expected DNA yield is 3-6 μg per ml. Routinely performed in most labs

MINIPREP EXTRACTS PLASMID

WHY MINIPREP? Need to purify to be able to sequence Need to purify to be able to clone Need to purify to be able to digest and run on a gel (size insert) Need to purify to transform Screen many plasmids for desired DNA fragment

PURPOSE OF SOLUTION I Glucose helps maintain osmolarity & Tris is used to buffer pH of suspension EDTA chelates divalent cations (ions with a 2+ charge) Chelating Mg++ destabalizes the bacterial cell membrane and inhibits the action of DNAses that would destroy DNA Rnase destroys the large quantity of RNA in a cell.

PURPOSE OF SOLUTION II NaOH-Loosens cell wall and releases DNA, Denatures chromosomal DNA through linearization and separation (does not affect plasmid DNA) SDS-creates holes in cell membrane and denatures proteins Viscosity due to denatured chromosomal DNA Green=proteins Red=Chromosomal & plasmid DNA Blue=RNA

PURPOSE OF SOLUTION III Sodium acetate-neutralizes NaOH Chromosomal DNA tries to renature at neutral pH but inefficient because completely separated due to its linear nature Salt ions-aggregate protein –SDS complex causing them to precipitate. Chromosomal DNA gets trapped in precipitate before it can renature. Plasmids able to renature and remain soluble

PURPOSE OF WASH BUFFER 80% ethanol to wash contaminates away from DNA Also contains Tris to buffer solution Also contains EDTA which chelates any metals that can be used by nucleases to degrade plasmid DNA Skipping this step will result in useless impure plasmid DNA

Method 13,000rpm 1min Discard the sup Add 200 of FAPD1 Buffer & resuspend the pellet Add 200 of FAPD2 Buffer & inverting 10 times Incubate at RT for 2min(Do not proceed this step over 5min) Add 300 of FAPD3 Buffer & inverting 10 times Centrifuge 5min Transfer the sup to FAPD Column & centrifuge 30sec Add 400 of W1 Buffer & centrifuge 30sec Add 600 of Wash Buffer & centrifuge 30sec Idle 3min Place FAPD Column to new tube Add 50~100㎕ of T.D.W Stand the column for 2min Centrifuge 1min Store plasmid DNA at -20