Fescue Chloroplast Genome Project - Part III
Plasmid Extraction and Restriction Digest
Plasmid Extraction
Goal – extract the plasmid DNA from a liquid bacteria culture.
Materials
 Resuspension Buffer
 Lysis Buffer
 Neutralization Buffer
 Glass-milk suspension
 Collection columns
Introduction
During the last lab you transformed bacteria with your newly constructed plasmid and
spread potential bacterial transformants onto LB medium with ampicillin, IPTG, and XGal. Bacteria that received the plasmid gained resistance to the antibiotic ampicillin and
grew into colonies on your plate. If the plasmid also contained the piece of DNA you
amplified during week one then the lacZ gene will have been interrupted and the bacteria
will be unable to convert X-Gal into a blue substrate.
My plate looks like this
During the last week a white colony was chosen from your plate and used to inoculate
liquid LB with ampicillin. This was cultured overnight and brought to your lab section.
Collect the culture tube labeled with your clone number.
We will use a commercial kit (Promega’s Wizard Miniprep) to extract and purify the
plasmid DNA from your bacterial culture. The procedure is a variation of the procedure
you followed during the first week of lab.
Wizard Miniprep Procedure
1. Pipette 2.0ml of culture into a microfuge tube.
2. Pellet cells for 1minute at full speed.
3. Remove and discard supernatant and Resuspend your pellet in 200μl
of Resuspension Solution by vortexing.
4. Add 200μl of Cell Lysis Solution and mix by rocking the tube in your
fingers.
This is the equivalent of the SDS buffer you added to your bacteria during the
first lab. It also contains NaOH to make the solution alkaline.
5. Add 200μl of Neutralization Solution and mix by rocking the tube.
A white precipitate should form.
The solution is Potassium Acetate. The acetic acid neutralizes the NaOH
rendering the SDS insoluble. When the SDS leaves solution it takes all the
denatured protein with it.
6. Centrifuge at full speed for 5 minutes.
The purpose of this step is to pellet the white precipitate of SDS, protein, and
cellular debris.
7. Label a syringe barrel with your clone number and
thread it onto a minicolumn on the vacuum manifold
by twisting clockwise until firmly seated.
8. Pipette the supernatant from step 6 into the syringe
barrel. Open the stopcock below your syringe and
allow the liquid to pass through the minicolumn.
Close the stopcock when liquid has completely
passed through.
Vacuum manifold
The minicolumn contains very tiny charged glass beads.
As the aqueous supernatant passes through the glass matrix
in the column, charged molecules (like DNA) will stick to
the beads.
9. Add 2ml of Wash Solution, open the stopcock
and allow the solution to pass through the minicolumn. After liquid has passed through leave
the stopcock open for 30 seconds (but no more)
to allow the vacuum to partially dry the resin.
Wash solution is a mixture of solutes and alcohol. This mix
contains just enough ethyl alcohol to prevent DNA from
solubilizing and leaving the glass bead matrix. Almost all
other charged molecules will become soluble and wash off
of the glass matrix.
Syringe
Barrel
Minicolumn
Stopcock
10.Remove the syringe barrel from the minicolumn by twisting it
counter-clockwise. Remove the minicolumn by twisting counterclockwise and place it in a 1.5ml microfuge tube.
11.Centrifuge for 2mins at full speed to remove any residual wash buffer.
Transfer the column to a new microfuge tube and discard the one that
collected the residual buffer.
12.Add 50μl of sterile water to the top of the minicolumn and wait 1
minute.
Only plain water has a high enough charge to encourage the DNA to leave the
beads and enter solution.
13.Centrifuge at full speed for 20seconds.
The water in your tube should contain your plasmid DNA.
14.Remove and discard the minicolumn.
Restriction Digests
Goal – confirm that your plasmid contains an insert.
Materials
 Your plasmid
 Restriction endonuclease
 Restriction Buffer
Introduction
Now that you have plasmid DNA you want to confirm that it is the correct one.
It’s almost impossible to be 100% certain that your fragment is indeed the one in your
vector but you can do a simple test to at least insure that a fragment is present and that it
is a similar size to the one you intended to clone.
1. Your plasmid
carrying the intended
insert.
2. The EcoRI enzyme will cut
the cloning vector on either
side of the insert
3. The fragment has
been released from
the plasmid. At least
two pieces of DNA
will appear on the
gel next week if a
fragment was presnt
in your plasmid.
The cloning vector has been engineered to carry short sequences of DNA that are
recognized by endonucleases (enzymes that cut in the middle of a DNA strand) called
restriction enzymes. A restriction enzyme will usually only cut DNA when it recognizes
and binds to a specific sequence. For example, we will use the enzyme EcoRI which
only cuts the sequence 5’-cttaag-3’. The pGemT vector has 5’-cttaag-3’ on both sides of
the cloning site. If we treat your plasmid with the EcoRI enzyme it will cut on either side
of the inserted fragment making at least two pieces of linear DNA from one circular piece
(see figure above).
Procedure
1. Mix the following solutions in a microfuge tube.
15μl of enzyme mix = EcoRI
Reaction Buffer
Water
5μl your plasmid
2. Label the top of your microfuge with your clone number.
3. Place in the designated test tube rack.
4. Your instructor will incubate the reaction overnight at 37°C and will store the
reaction in the freezer until next week.
Lab 2120 Cloning Part III
Lab Report
Your bacterial culture was prepared using a white colony from last week’s plate. Why
was a white colony chosen?
Were you attempting to isolate all of the DNA present in the bacterial cells? Explain.
Why is ethyl alcohol present in the wash solution that is passed through the minicolumn?
Look back at the cloning vector map given to you as part of the last handout. Is there an
enzyme other than EcoRI which could cut on both sides of the fragment and release it?
Make a rough sketch of a gel showing the two fragments you expect from this digest.
How could more than two fragments be produced by the restriction digest?