Download Plasmid Minipreps

Plasmid Minipreps
Kits…
Mini-Prep:
A rapid, small scale method of obtaining or
retrieving plasmid DNA (plasmid DNA +
foreign/inserted DNA) from bacterial cells.
2 General Variations:
1. Boiling Method
2. Alkaline Lysis Method (With a
modification to collect plasmids)
Overall Goals:
To lyse cells using Alkaline Lysis
Method
To precipitate out proteins and bacterial
DNA using Sodium Acetate
To separate plasmid from solution with
a column.
 To freeze plasmid DNA for further study
Preparation. Grow the bacteria
Grow an overnight (ON) culture of the desired bacteria
in 2-5 ml of LB medium containing the appropriate
antibiotic for plasmid selection. Incubate the cultures
at 37°C with vigorous shaking.
p. 1-12
2a. Transfer the cells to a tube
and centrifuge
Transfer 1.5 ml of the culture to
a microfuge tube and pellet the
cells for 1 minute at full speed
(12,000 rpm) in the
microcentrifuge.
First tap or gently vortex the glass
culture tube to resuspend the cells
which have settled. The culture can
be transferred to the microfuge
tube by pouring.
(Follow steps in Lab 6)
p. 1-12
2b. Remove the supernatant
Remove the growth medium (supernatant or
sup) by aspiration or by using the P-1000.
Leave the bacterial pellet as dry as possible so
that additional solutions are not diluted.
p. 1-12
Important Solutions and Their Purpose:

Solution 1: GTE (Glucose Tris EDTA):
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Tris is a buffer that works at physiological pH
(pH = 7.4).
EDTA binds divalent cations in the lipid bilayer,
thus weaking the cell membrane and also
protecting DNA from DNAses by removing
their cofactors.
Solution 2: SDS/NaOH (Sodium
dodyclsulfate + Sodium Hydroxide) - Alkaline
Lysis Solution:
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The detergent SDS(Soap) dissolves the lipid
component of the cell membrane (plasma
membrane), as well as cellular proteins
The NaOH denatures the chromosomal and
plasmid DNA into single strands (ssDNA). The
intact circle of plasmid DNA remain interwined.
Important Solutions and Their Purpose:

Solution 3: KOAc (Potassium Acetate + Acetic
Acid):
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The Acetic Acid returns the pH to neutral, allowing DNA strands to
renature. The large, disrupted chromosomal DNA strands can not
rehybridize perfectly, but instead collapse into a partially hybridized
tangle.
At the same time, the Sodium Acetate precipitates the SDS from
the cell suspension along with proteins and lipids with which it has
associated. The renatured chromosomal DNA is trapped in the
SDS/Lipid/Protein precipitate. Only smaller plasmids DNA and
RNA molecules escape the precipitate and remain in solution.
2
2.
3.
Important Solutions and Their Purpose:
Wash solution
-note the smell of alcohol (it is 80%
Ethanol)
-Washes through contaminants (ie. small
RNAs while the plasmid DNA stays
bound to the column's silica filter.
Spin
FYI: if doing “old fashion” mini prep w/ no
column, you must add Rnases to the
protocol
Discard
“flow through”
that contains
contaminants
Important Solutions and Their Purpose:
Drying step
-Is essential to remove as much of the
Ethanol as possible
If skipped many complications for use of plasmid
later. For example:
-can not run plasmid on gel for basic analysis
-can not sequence Plasmid DNA
Spin
-DO NOT SKIP!!!
Discard
“flow through”
that contains
contaminants
Important Solutions and Their Purpose:
Elution Solution. This low salt
buffer will cause the plasmid
to release from the column
and come off in the spin.
Switch out
collection tube for
1.5ml flip top MCT
Step by Step
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1. Spin the culture and remove LB broth
2. Resuspend in GTE
3. Lyse in Soap and Base (SDS/NaOH)
4. Neutralize to renature the plasmids
5. Spin to remove Genomic DNA and cell
debris.
6. Transfer Supernatant to column and spin
7. Wash by spining with Wash buffer
8. Elute and Collect Plasmids