Transcript MOLECULAR GENETICS

MINIPREP
WHAT IS A MINIPREP?
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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?
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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
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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
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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
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Sodium acetate-neutralizes
NaOH
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Chromosomal DNA tries to
renature at neutral pH but
inefficient because completely
separated due to its linear nature
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Salt ions-aggregate protein –SDS
complex causing them to
precipitate.
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Chromosomal DNA gets trapped
in precipitate before it can
renature.
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Plasmids able to renature and
remain soluble
PURPOSE OF WASH BUFFER
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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)
13,000rpm 1min
2)
Discard the sup
3)
Add 200 of FAPD1 Buffer & resuspend the pellet
4)
Add 200 of FAPD2 Buffer & inverting 10 times
5)
Incubate at RT for 2min(Do not proceed this step over 5min)
6)
Add 300 of FAPD3 Buffer & inverting 10 times
7)
Centrifuge 5min
8)
Transfer the sup to FAPD Column & centrifuge 30sec
9)
Add 400 of W1 Buffer & centrifuge 30sec
10)
Add 600 of Wash Buffer & centrifuge 30sec
11)
Idle 3min
12)
Place FAPD Column to new tube
13)
Add 50~100㎕ of T.D.W
14)
Stand the column for 2min
15)
Centrifuge 1min
16)
Store plasmid DNA at -20