MINIPREP WHAT IS A MINIPREP A plasmid DNA

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

PURPOSE OF SOLUTION I Glucose helps maintain osmolarity & Tris is used to buffer p. H 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 Na. OH-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 Na. OH Chromosomal DNA tries to renature at neutral p. H 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 1) Sample 1. 5 ml transfer, 13, 000 rpm 1 min CNFG 2) Discard the sup 3) Add 200 of FAPD 1 Buffer & resuspend the pellet 4) Add 200 of FAPD 2 Buffer & inverting 10 times 5) Incubate at RT for 2 min(Do not proceed this step over 5 min) 6) Add 300 of FAPD 3 Buffer & inverting 10 times 7) Centrifuge 5 min at full speed 8) Transfer the sup to FAPD Column & centrifuge 30 sec and then discard the flow-through → place the FAPD column back in the collection tube 9) Add 400 of W 1 Buffer & centrifuge 30 sec and then discard the flow-through → place the FAPD column back in the collection tube 10) Add 600 of Wash Buffer & centrifuge 30 sec and then discard the flow-through → place the FAPD column back in the collection tube 11) CNFG 3 min to dry the column 12) Place FAPD Column to new 1. 5 ml tube 13) Add 50~100㎕ of T. D. W and stand the column for 2 min 14) Centrifuge 1 min 15) Store plasmid DNA at -20°C