LABORATORY Day 3: Isolation and Analysis of Recombinant
Plasmid DNAs Containing Cloned 16S
Sequences
Objectives of Laboratory 1D:
1. Purify plasmid DNA from liquid cultures of one white transformant
2. Cleave the purified DNA with the restriction endonuclease EcoRI
3. Analyze these DNAs using agarose gel electrophoresis
4. Observe your plates of cultured soil microbes
Flow Chart of Laboratory 1D:
Purify One
Plasmid DNA
Cleave DNA
With EcoRI
Analyze DNA using
Electrophoresis
Observe Cultured
Soil Microbes
I. INTRODUCTION: Earlier this week, you isolated metagenomic DNA from your soil
sample, used PCR to amplify 16S sequences from this sample and cloned them into pCR2.1.
Today, you and your partner will each isolate and analyze plasmid DNAs from one of your white
transformant before the purified DNAs are sent for nucleotide sequence determination.
A diagram of the vector pCR2.1, into which you cloned your amplified 16S genes, is shown above. Note
that the polylinker has two recognition sequences/sites (arrows above) for the restriction enzyme EcoRI
and that these two sites flank the region into which your fragment was inserted. Today, you will make use
of the fortuitous locations of these EcoRI sites to analyze the structure of your recombinant plasmids.
II. EXPERIMENTAL PROCEDURES: Today you will 
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use the QIAprep Miniprep kit to isolate plasmid DNA from one of
the cultures you inoculated yesterday. This kit is widely used in our
Department because it produces very pure yields of plasmid DNA
that are suitable for sequencing and is also fast and relatively simple.
A. Isolation of Recombinant Plasmid DNA: Each person will 
isolate plasmid DNA following the procedure described below.
1. Obtain the two tubes containing the cultures you inoculated  Choose one of these tubes to use in
isolating plasmid DNA.
yesterday from the incubator.
2. Obtain a clear 1.7 ml microtube from the canisters on the front 
bench and label the top with your initials and the number of the
culture you’ve chosen.
3. Use your P1000 to transfer 1.0 ml from your culture tube to the 
microtube.
4. Pellet the bacterial cells in this culture by spinning it at maximum  Be sure to place your tube directly
across the rotor from another tube
speed for 1 min.
for balance.
5. Pour off the clear supernatant above the pellet into the sink.

6. Vortex vigorously for about 30 sec to resuspend the pelleted cells 
in the residual liquid.
7. Add 250 l Buffer #1 (tube #1, purple dot) to your tube and mix by  Make sure no clumps of cells are
present.
vortexing vigorously.
8. Add 250 l Buffer #2 (tube #2, red dot) to your tube and mix  The solution will turn blue.
thoroughly by inverting the tube 4 – 6 times. Do not vortex.
9. Add 350 l Buffer #3 (tube #3, orange dot) to your tube and mix it  This buffer causes genomic DNA,
which has a large molecular
immediately and thoroughly by inverting it several times. The
weight, to precipitate. The plasmid
addition of Buffer #3 should make the solution colorless.
DNA, which is much smaller, stays
However, if any blue color or clumps remain, invert your tube to
in solution.
mix until they are no longer visible.
10. Centrifuge for 10 min at 13,000 rpm (maximum speed) in the  A large white pellet of genomic
DNA and debris should be visible.
microfuge.
11. Pour the supernatant (the solution above the pellet) in your tube  Do not discard the supernatant.
It contains your plasmid DNA.
into a QIAprep spin column.
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12. Centrifuge your column for 30 – 60 sec at 13,000 rpm and  Use 13,000 rpm for all remaining
centifugation steps.
discard the flow-through into the sink.
13. Wash your QIAprep spin column by adding 0.75 ml buffer #4 
(tube #4, blue dot) and centrifuging for 30 – 60 sec.
14. Discard the flow-though from your tube and centrifuge for an  Discarding the flow-through is
essential before spinning again.
additional 1 min to remove residual wash buffer.
15. Place your QIAprep column in a clean 1.5 ml microfuge tube 
and add 50 l sterile H2O (clear tube, blue dot) to the center of
the QIAprep column.
16. Let your column stand for 1 min and then spin for 1 min.

17. After the spin ends, discard the column and close the top of your
tube.
18. Spin your tube in the microfuge for a few seconds to bring the
DNA solution to the bottom of the tubes.
B. Cleavage of your Purified Plasmid DNAs with EcoRI: In order  The name EcoRI is derived from
the first letter of the Genus and first
to confirm that the plasmid DNA you have purified contains an
two letters of the species name of
inserted fragment before it is sent out for sequence analysis, you will
the source organism, which is E.
cut an aliquot of your plasmid DNA with the restriction
coli, strain RY12.
endonuclease EcoRI.
1. Obtain an Isotherm and some ice before beginning.

2. Take your Isotherm and ice to the front bench to obtain a  Each restriction endonuclease has a
specific buffer, which provides the
microtube containing EcoRI (pink tube in Stratacooler) and
correct reaction conditions required
another containing 10X EcoRI buffer (pink tube, black dot).
for its optimal activity.
3. Take these tubes to the microfuge and pulse spin them for a few 
sec to bring the contents to the bottom.
4. Use the plasmid DNA you just isolated to make the following  Label the top of each tube with the
abbreviation of each transformant.
digest in a small clear microtube by adding the components in the
order listed below:
13 l sterile water (clear tube, blue dot)
2 l 10X EcoRI buffer (pink tube, black dot)
 When adding small amounts of
3 l plasmid DNA (flick the tube to mix these components)
2 l EcoRI enzyme (pink tube)
20 l total volume
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components, add them to the liquid
already present in the tube and not
onto the side of the tube.
5. Flick your tube with your fingers to mix it well and pulse-spin it
in the microfuge.
6. Place your tubes in a foam “floatie” and incubate at 37o C for 30 -  Return the rest of your plasmid
DNA to the front lab bench so it
40 min.
can be sent out for sequencing.
7. During this incubation, pre-run an e-gel as described next.
C. Pre-running Your E-Gel: Eight or nine people will share one
1.2% e-gel to analyze the digests of their plasmid DNAs. Follow the
same procedure you have used previously (pp. 40 – 42 of this
manual) to pre-run your e-gel.
D. Loading and Running Your E-Gel: Use both hands to remove 
the gel comb by gently lifting it and rolling it slowly toward you.
1. Obtain a sample of the 123 bp Ladder (yellow tube) and a sample  Flick these tubes gently but
thoroughly to mix well.
of Lambda DNA cut with HindIII (clear tube, green dot) from the
front bench.
2. When the incubation is over, retrieve your tube and add 2 l 
loading dye (clear tube, purple dot) to it.
3. Flick your tube with your fingers to mix and pulse-spin it in the 
microfuge.
4. Load 20 µl of the 123 bp Ladder (yellow tube) into one end lane.

5. Load 20 µl of the Lambda DNA cut with HindIII (clear tube, green 
dot) into the other end lane.
6. Each person should load 20 l of her/his digest in one of the other  Be sure to record the number of
the gel and the lane you are using.
wells.
7. The gel will run for 20 -30 min until the dye has migrated slightly 
more than halfway and then you will take a picture for your
records.
E. Observing Your Cultured Soil Microbes: While your gel is  Record these numbers in your
notebook.
running, count any new colonies of soil microbes that have grown
since your last observation. Also take a digital picture of some or all
of your plates for your records.
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