1. MINIPREP

2. 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

3. MINIPREP EXTRACTS PLASMID

4. 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

5. 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.

6. 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

7. 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

8. 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

9. 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