MINIPREP & RESTRICTION
ENZYMES
WAKSMAN STUDENT
SCHOLARS PROGRAM
MARTY EDELBERG
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
MINIPREP STEPS
 Start with
overnight culture
of E. coli
containing
plasmid with DNA
insert of interest
 Tap the culture
tube to resuspend
cells and pour into
1.5 ml microfuge
tube.
MINIPREP STEPS-continued
 Spin microfuge in
microcentrifuge for 1
minute at 14,000 RPM
 Purpose-to form a
pellet (separate cells
from growth medium
and concentrate
cells)
 Removes cell waste
MINIPREP STEPS-continued
Remove
supernatant
by pouring or
pipetting
MINIPREP STEPS-continued
 Resuspend the
bacterial pellet by
adding 200 ul of
solution I
 Can vortex or
pipett up and
down to
resuspend
 Pellet should be
completely
dispersed.
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.
MINIPREP STEPS-continued
Add 200 ul of solution II
Mix gently by inverting 5 times
Note a viscous bacterial lysate
Aggressive mixing may shear
chromosomal DNA into small
fragments and contaminate
prep
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
MINIPREP STEPS-continued
Add 400 ul of solution III
Mix gently by inverting 10 times
Note a white precipitate
Aggressive shaking may break
chromosomal DNA and
contaminate prep
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
MINIPREP STEPS-continued
Centrifuge for five minutes at full
speed
A white precipitate will form on the
bottom and side of the tube
Plasmid DNA remains in
supernatant
Chromosomal DNA and proteins in
precipitate
MINIPREP STEPS-continued
 Pour the supernatant into an appropriately labelled
spin column that has been inserted into a collection
tube
 Avoid adding the white precipitate
 Incubate at room temperature for 1 minute
 Microcentrifuge for I minute at full speed
MINIPREP STEPS-continued
Pour off the
flow through
from the
collection
tube
MINIPREP STEPS-continued
 Add 400 ul of
wash buffer to
spin column
 Centrifuge at 1
minute at full
speed
 Pour off the the
flow through from
the collection
tube
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
MINIPREP STEPS-continued
Centrifuge the spin column again for 1
minute at full speed
Ensures removing any residual ethanol
Place spin column in fresh microcentrifuge
tube appropriately labelled
Failure to do this step will cause DNA to float
out of gel & give a poor sequence result.
MINIPREP STEPS-continued
Add 60 ul of elution buffer-removes
plasmid DNA from spin column
Incubate at room temperature for 1
minute
Centrifuge at full speed for I minute
Throw away spin column and keep
microfuge tube with liquid.
Store in freezer.
What tools do we use to cut DNA of interest and
join it to a plasmid or remove from plasmid?
Restriction enzymes
Proteins that cut DNA at defined
sequences 4-8 bp long called
restriction sites
Cut phosphodiester bonds that link
nucleotides together
Cut in a precise and predictable
manor, thus reproducible
Restriction fragments-piece of cut
DNA
Where do restriction
enzymes come from?
Example-EcoR1 restriction
enzyme
How are restriction enzymes
named?
EcoRI from Escherichia
coli
BamHI from Bacillus
amyloliqueraciens
PvuI and PvuII are different
enzymes from same strain.
Genus-species-strain-order of
discovery
What restriction enzymes do
we use in our research?
Sfi used to cut Duckweed DNA and
plasmid for joining
A.f.Insert
insert
PvuII-cuts insert out of
plasmid
Serve as landmarks in plasmid to help find
insert when editing sequence
Kpnl
SMA I
ECORI
ScifiA
XBA I
XHO I
HIND III
GGTACC
CCCGGG
GAATTC
GGCCATTACGGCC
TCTAGA
CTCGAG
AAGCTT
When we work with enzymes
must create optimal working
environment
Need buffer (pH, salt conc)
Proper temperature-KEEP ON ICE
Poor conditions may:
deactivate enzyme
cause starr activity
What is most important to
remember?
Always keep enzymes on ice
(denaturation)
Always use fresh tips
Keep record in log book
clone name
date of digest
DIGEST PROCEDURE
Label I microfuge tube 5X
Digest mix
Label ____ tubes with clone
name and digest (see how to
label next slide)
How do we label our
clones?
13ME01.09
13=PHHS
ME=Initial of person who made clone
01=Number assigned to clone
09=Year of project
DIGEST PROCEDURE
1 Reaction mix
dd H2O
7ul
10X buffer
2ul
Miniprep DNA 10ul
PvuII
1ul
5 reaction mix
dd H2O
35ul
10X buffer
10ul
Miniprep DNA *
PvuII
5ul
DIGEST PROCEDURE
 Mix reaction mix by pipeting up and
down
 Add 10 ul of reaction mix to each
microfuge tube labeled with a clone
name
 Add 10ul of the corresponding DNA to
the corresponding labeled tube
 Mix each tube by tapping or in centrifuge
at low for a few seconds
 Incubate for 1hour at 37C
 Add 2ul of 10X loading gel
 Store in freezer -20C