Genetics 3250 Sequence Analysis
Cahoon – Genetics 3250 Lab
Middle Tennessee State University
Chloroplast Genome Project – Week 1
In plant cells, DNA can be found in three organelles – nuclei, chloroplasts, and
mitochondria.
chloro
plast
nucleus
mitochondrion
mitochondrion
chloro
plast
chloro
plast
The chloroplast is the site of photosynthesis in plant cells. It’s
genome and its process of gene expression is very similar to those
found in bacteria.
Maier et al. 1995,
J. Mol. Biol
251:614-628
The chloroplast genome can be imagined as a circular piece of
DNA about 120,000 – 140,000 bases in size with approximately
100 genes. This diagram represents the corn chloroplast genome
which was completed in 1995.
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Genetics 3250 Sequence Analysis
Cahoon – Genetics 3250 Lab
Middle Tennessee State University
You and your genetics class colleagues are attempting to sequence the chloroplast
genome of tall fescue (Festuca arundinacea).
Intact genomes are generally too large to sequence. To get around this problem a genome
must be cut into lots of small manageable pieces. This semester, two independent study
students have isolated the chloroplast genome, mechanically sheared the DNA into small
pieces and then placed them into plasmid cloning vectors for sequencing.
genome
Shearing
force
Genome cut into
thousands of tiny
random pieces
Pieces
put into
plasmid
vectors
These plasmids are then grown inside bacteria that will produce the millions of copies
that are required for manipulation and sequencing. Today you will extract a plasmid
from a bacterial culture. Next week we will use this plasmid to set up a sequencing
reaction.
Plasmid DNA Extraction
Plasmid Extraction
Goal – extract the plasmid DNA from a liquid bacterial culture.
Materials
 Resuspension Buffer (P1)
 Lysis Buffer (P2)
 Neutralization Buffer (N3)
 Wash Buffer (PE)
 Spin Column
 Elution Buffer (EB)
 Collection tubes
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Genetics 3250 Sequence Analysis
Cahoon – Genetics 3250 Lab
Middle Tennessee State University
Introduction
Today you will use a commercial kit (Qiagen’s Miniprep) to extract and purify plasmid
DNA from your bacterial culture. Plasmids are small circular pieces of DNA that are
maintained and replicated separately from the bacterial cell’s genome.
Procedure
1. Pipette or pour 2.0ml of culture into a colored microfuge tube.
2. Pellet cells for 1minute at full speed.
3. Pour off the supernatant (growth medium) and blot the open tube
against a paper towel. Resuspend your pellet in 250μl of
Resuspension Solution (P1) by vortexing until you no longer see a
pellet.
This step removes the salty growth medium and resuspends in a buffered
solution which will aid the extraction.
4. Add 250μl of Cell Lysis Solution (P2) and mix by rocking the tube in
your fingers. The solution should become mostly clear and viscous.
The cell lysis solution contains a strong detergent (Sodium Dodecyl Sulfate) and
base (Sodium Hydroxide) which will lyse the cells and denature proteins and
membrane components so the cell will spill its contents. After this step you have
a mixture of every type of molecule found in the cell. The challenge is to purify
the DNA away from everything else (protein, lipid, carbohydrate, and insoluble
debris).
5. Add 350μl of Neutralization Solution (N3) 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 most of the
protein and lipid with it.
6. Centrifuge at full speed for 10 minutes.
The purpose of this step is to pellet the white precipitate of SDS, protein, and
cellular debris.
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Genetics 3250 Sequence Analysis
Cahoon – Genetics 3250 Lab
Middle Tennessee State University
7. Label a minicolumn with your clone number.
Pipette the supernatant from step 6 into column. Centrifuge for 30-60
seconds. Remove the column, pour off the flow-through and replace
the column in the tube.
The minicolumn contains microscopic charged glass beads. As the aqueous
supernatant passes through the glass matrix in the column, certain hydrostatic
molecules (like DNA) will stick to the beads. Most of the residual proteins and
carbohydrates will pass by the beads and end up in the flow-through which is
discarded.
8. Add 750 μl of Wash Solution (PE) to the column and centrifuge for
30-60 seconds. Remove the column, discard the flow-through and
replace the column in the tube.
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.
9. Centrifuge the column in the collection tube for 1 minute to remove
any residual wash buffer.
10.Label a new microcentrifuge tube with your clone number and name.
Place the column into the new tube.
11. Add 50μl of Elution Buffer (EB) or Sterile Water to the column.
Let stand for 1 minute and then centrifuge for 1 minute.
The interaction between DNA and water is stronger than the interaction of DNA
with the beads so it is pulled away and ends up in your flow through.
12.THE FLOW THROUGH CONTAINS YOUR DNA
Save the flow through in the microcentrifuge tube.
Remove and discard the minicolumn.
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