Mini Prep

GTC Lab 4
GTC LAB 4: Plasmid Isolation

To analyze the DNA that you have cloned onto the pENTR vector, it is necessary to first
extract the plasmids from the E. coli cells. The plasmids may then be analyzed by restriction
enzyme digest. Many methods have been developed to purify plasmids from bacteria. All
methods involve three steps:
1. growth of the bacterial culture,
2. harvesting and lysis of the bacteria,
3. purification of the plasmid DNA
Growth of the Bacterial Culture
Plasmids should be purified from bacterial cultures that have been inoculated with a single
transformed colony picked from an agar plate. At all times, the transformed bacteria should be
grown in selective conditions, i.e., in the presence of appropriate antibiotic.
Harvesting and Lysis of the Bacteria
Bacteria are recovered by centrifugation and lysed by a number of methods, including heat,
alkali, and detergents, and some other gentler means. The choice of methods is decided based
on the strain of E. coli, the size of the plasmid, and the techniques used to purify the plasmid.
Smaller plasmids (<15 kb) are fairly durable, and can withstand more severe methods of lysis
such as those listed above. Treatment of bacterial suspensions with detergent or heat disrupts
the base pairing and causes the linear stretches of sheared chromosomal DNA of the host to
denature and fall away from each other. The strands of the plasmid DNA, however, are unable
to separate from each other because they are topologically intertwined! Returning the
conditions back to normal then allows the two strands to fall perfectly back into alignment and
renature into superhelical plasmid molecules.
In this lab, you will lyse the cells with base combined with the anionic detergent sodium
dodecyl sulfate (SDS). This combination has been used for more than 20 years for plasmid
isolation. Exposure of bacterial suspensions to the strongly anionic detergent at high pH opens
the cell wall, denatures chromosomal DNA and proteins, and releases plasmid DNA into the
supernatant. During lysis, the cell debris (broken cell walls, proteins, and denatured
chromosomal DNA) forms large complexes that are coated with SDS. These complexes fall
out of solution (precipitate) when sodium ions are replaced by potassium ions.
Purification of the Plasmid DNA
All methods of lysing bacteria will yield plasmid solutions contaminated with chromosomal
DNA and RNA. Centrifugation removes the vast majority of chromosomal DNA (it will form
a pellet, while plasmid DNA remains soluble), and treatment with RNase will eliminate
contaminating RNA.
GTC Lab 4
Procedure: There are three parts to this lab:

1) plasmid purification
2) plasmid analysis by restriction enzyme digestion
3) recombination of a correct clone into the GFP vector
Part 1: Recombinant plasmid purification
Day before lab:
A. Grow overnight cultures of transformants.
1. The day before lab, pick 4 transformants from each ligation with separate sterile
toothpicks, and inoculate separate culture tubes containing liquid medium (LB+Kan).
2. Grow overnight with shaking at 37C.
Day of lab:
B. Harvest and lyse bacteria.
1. Transfer 1.4 mL of bacterial cultures to a sterile microcentrifuge tubes.
Mix each culture well before taking the cells. Many cells will have sunk to the
bottom of the tube.
2. Centrifuge for 30 seconds at maximum speed.
3. Pour off the supernatant into a waste beaker by the centrifuge.
4. Resuspend the pelleted bacterial cells in 250 L of Buffer P1.
No cell clumps should be visible after resuspension of the pellet. This is a
glucose solution that adjusts the osmolarity of the cell suspension to
facilitate cell lysis. This buffer also contains RNase.
5. Add 250 L of Buffer P2. This is the alkaline SDS solution for cell lysis.
Invert the tube immediately (and gently) 10 times. DO NOT VORTEX!
The solution should become viscous and slightly clear. Invert the tube a
few more times if necessary to achieve this. WORK QUICKLY: Do not
allow the lysis reaction to go for more than 5 minutes or plasmid DNA
will be irreparably damaged (nicked!).
6. Add 350 L of Buffer N3 and mix by inverting several times.
This is a potassium acetate buffer that will cause precipitation of cell debris
including chromosomal DNA. You should see the solution immediately turn
cloudy. This may sit on ice until you are ready to centrifuge (next step).
C. Purify the plasmid DNA.
7. Centrifuge for 10 minutes at max speed. A white pellet will form.
8. Apply the supernatant to a plasmid prep column by decanting, or using a pipet.
Your plasmid DNA will be retained on the silica-gel membrane inside due
to the high salt conditions of the supernatant. The silica-gel binds only
dsDNA, while RNA and proteins are not retained, and instead come off in
the flow-through.
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GTC Lab 4
9. Centrifuge for 30 60s. Discard the flow-through.
10. Wash the bound plasmid DNA by adding 0.75 mL of Buffer PE and
centrifuging for 30 60 s.
This step is necessary to remove salts from the plasmid DNA. Salts can inhibit
restriction endonuclease digestions.
11. Discard the flow-through, and centrifuge for an additional 1 min to remove
residual wash buffer.
Buffer PE contains ethanol. Thus, it is very important to remove any residual
buffer as ethanol will inhibit subsequent enzymatic reactions.
12. Place the column in a clean microcentrifuge tube. To elute DNA, add 50 L
Elution Buffer (EB) or water to the center of each column, let it stand
for 1 min, and then centrifuge for 1 min.
DNA is released from the silica-gel membrane by its low salt content. This
buffer is 10 mM Tris, pH 8.5.
Part 2: Recombinant plasmid analysis by restriction enzyme digestion
1. Set up your analytical restriction enzyme digestions for each plasmid.
Use ~1 g of DNA in each reaction. Assume the concentration of plasmid DNA in your
purifications is ~0.2 g/L.
Using the buffer chart, determine which NEBuffer to use.
Make a master mix for 5 reactions of all components except plasmid. Pipet the
appropriate amount into 4 microcentrifuge tubes leaving room to add plasmid.
Restriction enzyme digest for ONE sample:
x 5 rxns:
___ L of plasmid prep DNA
___ L
___ L of 10X digestion buffer (from New England Biolabs, NEBuffer)
___ L
___ L of 10X BSA (some enzymes do not require this see buffer chart)
___ L
0.5 L of enzyme (of each enzyme if you are doing a double digest with two enzyme)
___ L sterile distilled MilliQ water (to 20 L total reaction volume)
20 L total volume
2.5 L
___ L
100 L
GTC Lab 4
2. Incubate the reactions at 37C for two hours. Add 2 L of 10X loading buffer to the digest
and load 20 L onto a 0.8% agarose gel containing EtBr (0.5 L). Electrophorese at 100 V for
45 minutes. Load 5 L of 1 kb ladder in one lane.
Part 3: Recombination into GFP vector
The L-R recombination reaction
The LR Clonase enzyme is a purified form of phage Lambda recombinase. It will remove the
coding sequence from the pENTR/D-TOPO vector and replace the gateway cassette in the new
pIGF-GTW vector for transformation into Tetrahymena. Recombining your cloned gene into
this new vector will create an in-frame fusion of your gene with GFP. Its expression will be
under the control of the MTT promoter, which is inducible with divalent metals such as
cadmium.
SettinguptheLRrecombinationreaction
LR Clonase II enzyme mix is supplied as a 5X solution.
1. Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix:
1-3 l of pENTR plasmid clone (50-150 ng total)
1 l of pIGF-GTW destination vector (400 ng/l)
H2O to 4 l
2. To each reaction above, add 1 l of LR ClonaseII enzyme and mix well by pipeting up
and down being careful not to introduce air bubbles.
3. Incubate reactions at room temperature overnight.
Your instructor will store them at -20C the next day.
GTC Lab 4
GTC LAB 4 INSTRUCTORS GUIDE

Prerequisite information: Restriction enzymes; enzyme recognition sites
Students will gain:
understanding and practice applying molecular /biochemical principles underlying nucleic
acid isolation
practice setting up basic molecular enzymatic reactions
Time: Approximately 4 hrs
Materials:
Qiagen plasmid miniprep kit

NotI restriction enzyme plus an additional variety of other restriction enzymes
restriction enzyme buffers
Clonase enzyme kit (Invitrogen)
400 ng/L prep of destination vector
37C water bath and tube floaters
agarose (molecular biology grade)
1X TAE running buffer
EtBr (10mg/mL solution)
microcentrifuge tubes
gel running apparatuses
power supplies
UV light board and digital camera
General
Part 1: Plasmid isolation
Aliquot the plasmid isolation buffers from the kit so that there are 3-4 sets available for
students to use.
Encourage students to mix their bacterial cultures well before taking 1.4 mLs. Many cells
will have settled to the bottom of the culture tubes.
Part 2: Restriction Digestion
It saves time if students come to lab prepared with knowing which enzymes they will
use for restriction digestion. The enzymes they use should cut once (or twice) in the
vector and once (or twice) in their cloned piece of DNA such that it will yield a banding
pattern allowing them to distinguish whether their gene was cloned, and in which
direction it was inserted. This is a good prelab assignment for them to figure out. A
restriction map of the pENTR vector is included. This may be copied and given to
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GTC Lab 4
students in advance. They should already have restriction sites mapped on their gene
from the bioinformatics module.
If no restriction sites can be found for the available enzymes, digest with NotI (cuts
once in the vector) and students will be able to see if the shift in band size is consistent
with that expected for cloning the correct sequence. It will not yield information on
direction, but only a small fraction of inserts go in the wrong direction.
Digesting 5 L of each plasmid preparation should be sufficient.
While the restriction digests are incubating, students should pour their agarose gels so that
they are ready to load as soon as the digest is complete.
Part 3: Recombinase (Clonase) reaction

The pIGF-GTW destination vector should be of high quality: supercoiled, RNA and proteinfree.
To get high yields of the destination vector, try isolating vector from E coli using the boiling
protocol in GTC 6. The vectors are generally too large to be isolated using conventional
column methods.
The destination vectors can be phenol:chloroform extracted and RNase treated.

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GTC Lab 4

GTC LAB 4: Plasmid Isolation

Procedure: There are three parts to this lab:

Using the buffer chart, determine which NEBuffer to use.

Restriction enzyme digest for ONE sample:

___ L of plasmid prep DNA

___ L of 10X digestion buffer (from New England Biolabs, NEBuffer)

GTC LAB 4 INSTRUCTORS GUIDE

Qiagen plasmid miniprep kit

Part 2: Restriction Digestion

Digesting 5 L of each plasmid preparation should be sufficient.

Part 3: Recombinase (Clonase) reaction

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