DNA CLONING AND GENE EXPRESSION
INTRODUCTION
Recombinant DNA technology is at the heart of the biotechnology industry. In this lab, we will be
performing restriction enzyme cloning to create a new (recombinant) plasmid. It is this same method that
Herbert Boyer and Stanley Cohen used in 1973 to herald in the field of genetic engineering. The only
difference is that we will be using updated techniques and materials, ones that you will find in any
molecular biology laboratory today! We will also be expressing the gene we are cloning and purifying it
by column chromatography. Altogether, we will be performing the steps a molecular biologist would
carry out in engineering and production a biotechnological product.
We will be taking a gene of interest (GFP) and moving it from one plasmid to another. GFP will be
coming out of a plasmid containing the kanR gene which supplies the bacteria with resistance to
kanamycin. We will call this plasmid pKAN-GFP. GFP will be moved into an ampicillin resistant
plasmid called pGEM. Our final product should be pGEM-GFP. pGEM not only has an ampR gene, but
the lacZ gene as well. lacZ encodes for the enzyme β-galactosidase. This enzyme is normally used by
bacteria to cleave disaccharides, such as lactose, for metabolism. In this lab, we are going use βgalactosidase activity to track whether we have cloned GFP into pGEM successfully. Since we will clone
GFP into restriction sites in lacZ, a successful cloning event should disrupt its function. So we will be
engineering a plasmid that would allow cells to be ampicillin resistant, green, and have disrupted βgalactosidase activity.
β-galactosidase activity will be tracked by enzymatic cleavage of an artificial disaccharide, X-Gal. X-Gal,
when cleaved, will release a blue color. X-Gal and IPTG (lacZ is connected to the lac operon and must be
induced) will be plated with transformed cells, and successful pGEM-GFP transformants will not be blue,
while transformants with intact pGEM plasmids will turn blue. This typical selection procedure is called
blue/white selection and is a commonly used marker for successful recombination. Because our gene of
interest is also expressing its own color, we will be tracking three different colored colonies: blue, white,
and green!
The reason for such an elaborate tracking system is because so many things can go wrong! First,
fragments produced by restriction digestion can recombine randomly, as long as sticky ends are
compatible. Therefore, various combinations of fragments can come together and become ligated into a
new plasmid creating a large number of possible clones. Second, restriction digests do not always cut
every piece of DNA. Plasmids that happen to be cut by only one of the enzymes will probably re-ligate
onto itself. Plasmids that are not cut at all will not undergo any changes, but still be transformed. By
understanding what the different parts of the plasmids are doing, we can predict what the final
recombined plasmids will look like based on the phenotype of the transformed colonies.
Maps of the two original plasmids are shown on the next page. Restriction sites for a few select enzymes
are shown. Total sizes are in the center of the maps and cut sites are displayed in parentheses next to the
restriction enzyme.
CLONING AND GENE EXPRESSION
Ori
XhoI (2)
EcoRI (82)
BamHI (112)
Ori
pKAN-GFP
(2987)
KanR
pGEM
(3197)
GFP
PstI (865)
HindIII (873)
AmpR
lacZ
EcoRI (5)
BamHI (26)
SalI (38)
PstI (48)
HindIII (56)
The restriction enzymes we will use to cut out GFP are PstI and XhoI. pGEM will be cut with PstI and
SalI. PstI sticky ends will bind to PstI sticky ends. XhoI and SalI produce the same sticky ends and will
allow GFP to be cloned into pGEM. BamHI and HinDIII will be used in the analysis section to check the
correctness of the clones. This will involve the same kind of analysis performed in the GFP lab. A table of
the restriction cut sites of enzymes we will use in this lab is shown below.
Table 1. Restriction enzyme recognition (↓ indicates cut site)
BamHI
5’ G↓GATCC 3’
EcoRI
5’ G↓AATTC 3’
HindIII
5’ A↓AGCTT 3’
PstI
5’ CTGCA↓G 3’
SalI
5’ G↓TCGAC 3’
XhoI
5’ C↓TCGAG 3’
The GFP protein will be purified using hydrophobic interaction chromatography (HIC). The following is
information from Bio-Rad Inc.
GFP has several stretches of hydrophobic amino acids, which results in the total protein being
very hydrophobic. When the supernatant, rich in GFP, is passed over a HIC column in a highly
salty buffer (Binding Buffer), the hydrophobic regions of the GFP stick to the HIC beads. Other
proteins which are less hydrophobic (or more hydrophilic) pass right through the column. This
single procedure allows the purification of GFP from a complex mixture of bacterial proteins.
Loading the GFP supernatant onto the chromatography column
When students load the GFP supernatant onto their columns, it is very important that they do not
disturb the upper surface of the column bed when performing the chromatography procedure. The
column matrix should have a relatively flat upper surface. A slightly uneven column bed will not
drastically affect the procedure. However, subsequent steps of loading, washing, and eluting
should minimize disrupting the column such that beads "fluff up" into the buffer. When loading
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CLONING AND GENE EXPRESSION
the GFP supernatant onto the column, the pipette tip should be inserted into the column and should
rest against the side of the column. The supernatant should be slowly expelled from the pipette,
down the walls of the column. When the supernatant has completely entered the column, a green
ring of fluorescence should be visible at the top of the bed when viewed with the UV light. There
are four different buffers which are used in the HIC procedure. Each buffer should be slowly
pipetted down the side of the column to minimize disturbance to the column resins.
Equilibration buffer
A medium salt buffer (2 M (NH4)2SO4) which is used to "equilibrate" or "prime" the
chromatography column for the binding of GFP.
Binding buffer
An equal volume of high salt Binding Buffer (4 M (NH4)2SO4) is added to the bacterial lysate.
The end result is that the supernatant containing GFP has the same salt concentration as the
equilibrated column. When in a high salt solution, the hydrophobic regions of proteins are more
exposed and are able to interact with and bind the hydrophobic regions of the column.
Wash buffer
A medium salt Wash Buffer (1.3 M (NH4)2SO4) is used to wash weakly associated proteins from
the column; proteins which are strongly hydrophobic (GFP) remain bound to the column. When
the Wash Buffer is applied to the column, care should again be taken to minimize disturbance of
the column resin. Slow, steady pipetting down the side of the column is most effective. At this
point "a ring" of GFP should begin to penetrate the upper surface of the matrix (~ 1–2 mm into the
bed).
Elution buffer
A low salt buffer (TE Solution; 10 mM Tris/EDTA) is used to wash GFP from the column.
In low salt buffers (which have a higher concentration of water molecules), the conformation of
GFP changes so that the hydrophilic residues of GFP are more exposed to the surface, causing the
GFP to have a higher affinity for the buffer than for the column, thereby allowing the GFP to wash
off the column. The 0.75 milliliters of TE (Elution) Buffer is applied to the column gently, as
described above. The TE Solution will disrupt the hydrophobic interactions between the GFP and
the column bed, causing GFP to let go and "elute" from the column. The GFP should pass down
the column as a bright green fluorescent ring. This is easily observed using the UV light. If the
column bed was disturbed in any of the preceding steps, the GFP will not elute as a distinct ring,
but will elute with a more irregular, distorted shape. However, elution should still occur at this
step. If successful, collection tube 3 should fluoresce bright green.
This entire lab will take 5 lab periods. A summary of the lab activities is shown in the grid below.
Day 1
Restriction digest and ligation
Day 2
Transformation and plating
Day 3
Analysis of plates and preparing overnight cultures
Day 4
Miniprep and cell lysis for chromatography
Day 5
Restriction analysis and chromatography
On Day 1, a double digest using restriction enzymes that will cut out GFP and open up pGEM will be
performed. This will create four different fragments, each having sticky ends that can recombine with
complementary sequences. The fragments will be allowed to randomly recombine and their
recombination made permanent by ligation.
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CLONING AND GENE EXPRESSION
On Day 2, the recombinant plasmids will then be used to transform E.coli bacterial cells. Transformed
cells will be screened for resistance to each ampicillin and kanamycin and both together. X-Gal and IPTG
will also be added to appropriate plates.
On Day 3, plates will be removed from the incubator and analyzed. A blue, green, and white colony will
be picked from the ligation amp plate. Overnights will be prepared for each colony for minipreps.
On Day 4, minipreps of the three overnights will be performed. Green cultures will be lysed for
chromatography on Day 5.
On Day 5, restriction analysis of the DNA clones will be performed. Gel electrophoresis and staining will
be done. While gels are running, HIC will be performed.
DAY 1: RESTRICTION DIGEST
MATERIALS
0.075 µg/µl pKAN-GFP
0.075 µg/µl pGEM
2X restriction buffer
pKAN-GFP enzyme mix (PstI and XhoI)
pGEM enzyme mix (PstI and SalI)
Micropipets
PROCEDURE
1. Use Table 2 as a guide to prepare the tubes. Use a fresh tip for each reagent.
Table 2. Preparing restriction digest
Tube
pKAN-GFP
pGEM
2X Buffer
Enzyme mix
pAMP
5.5 µl
7.5 µl
2 µl
pKAN
5.5 µl
7.5 µl
2 µl
2. Add 7.5 µl of 2X restriction buffer to each tube
3. Use a fresh tip and add 2 µl of the appropriate enzyme mix to each tube.
4. Close caps, mix, and pulse in centrifuge.
5. Incubate at 37 °C for 30 minutes.
LIGATION OF RESTRICTION FRAGMENTS
MATERIALS
Digested pKAN-GFP and pGEM
2X ligation buffer/ATP
DNA ligase
Sterile microfuge tube
PROCEDURE
1. Incubate pKAN-GFP and pGEM digests at 65°C for 10 minutes. This step is necessary denature
restriction enzymes.
2. Use the table below as a guide to prepare the tubes. Use a fresh tip for each reagent.
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CLONING AND GENE EXPRESSION
Tube
Ligation
Digested
pKAN-GFP
3 µl
Preparing tubes for ligation
Digested
2X Ligation
pGEM
Buffer/ATP
3 µl
10 µl
Water
DNA Ligase
2 µl
2 µl
3. Add 3 µl of digested pKAN-GFP to the ligation tube .
4. Use a fresh tip and add 3 µl of digested pGEM to the ligation tube.
5. Use a fresh tip and add 10 µl of 2X ligation buffer/ATP to the tube.
6. Use a fresh tip and add 2 µl of DI water to the tube
7. Use a fresh tip and add 2 µl of DNA ligase to the tube. Suction in and out to ensure complete transfer.
8. Pulse tube in centrifuge. Incubate 24 hours at room temperature. (Refrigerate until next lab period)
DAY 2: TRANSFORMATION
Ligated DNA from the previous day will be used to transform JM109 E. coli cells. Purified pKAN-GFP
and pGEM plasmids will also be used as controls. Transformed cells will be plated onto 4 types of LB
agar: with ampicillin (LB/amp); with kanamycin (LB/kan); with both ampicillin and kanamycin
(LB/amp+kan); and with plain LB (LB).
PREPARING COMPETENT CELLS
All of the steps in this lab must be prepared under sterile conditions.
MATERIALS
2 microfuge tubes of mid-log E. coli (JM109) cells
Sterile CaCl2 solution
Micropipets and sterile tips
Beaker of ice
PROCEDURE
Disinfect workspace.
1. Place sterile CaCl2 solution on ice.
2. Obtain two microfuge tubes of mid-log JM109 cells. There should be about 1.5 ml in each tube.
3. Make sure caps are securely closed and place tubes in microfuge and spin at top speed for 2 minutes.
Ensure proper balance in rotor head before starting.
4. Carefully and sterilely pour off supernatant from each tube into your biohazard waste container. Make
sure tube is well drained by gently tapping on a small piece of clean paper towel (dispose of in
hazardous waste), but don't lose the pellet.
5. Sterilely add 500 µl of fresh, ice cold CaCl2 to each tube.
6. Pipette the pellet in and out to thoroughly mix the cells into the CaCl2. Hold tube up to the light to
make sure all the cells are mixed into solution.
7. Mix the contents of one tube and transfer all of its contents into the other tube (pool the competent
cells into one tube). You should have a total of 1000 µl of cells in one tube.
8. Return tube to ice and prepare for transformation step.
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CLONING AND GENE EXPRESSION
TRANSFORMATION
MATERIALS
pKAN-GFP (0.075 µg/µl)
pGEM (0.075 µg/µl)
X-Gal (25 µg/µl)
IPTG (12.5 µg/µl)
3 sterile microfuge tubes
Beaker of ice
Micropipets and tips
3 LB/amp+kan, 3 LB/amp, 3 LB/kan, 3 LB agar plates
Spreader, dish, and 95% ethanol
PROCEDURE
1. Disinfect workspace.
2. Label 3 sterile microfuge tubes as follows:
+pLIG = this tube will contain your ligated recombinant plasmids from the previous lab.
+pKAN-GFP = this will be a control tube containing pKAN-GFP.
+pGEM = this will be a control tube containing pGEM.
3. Sterilely add 200 µl of competent cells to each tube.
4.
Place all 3 tubes on ice.
5. Sterilely add 10 µl of your ligated recombinant plasmids to the "+pLIG" tube.
6. Use a fresh tip and sterilely add 10 µl of pKAN-GFP (0.075 µg/µl) to the " pKAN-GFP " tube.
7. Use a fresh tip and sterilely add 10 µl of pGEM (0.075 µg/µl) to the " pGEM " tube.
8. Close caps and gently mix. Avoid bubbles and getting the solution on the sides of the tube.
9. Return tubes to ice for 20 minutes.
10. During the incubation, obtain the 12 plates (3 each). Mark them as indicated in Table 3.
11. Carry the ice beaker to the 42°C water bath.
12. Heat shock the cells by immersing the tubes in the 42°C water bath for exactly 90 seconds.
13. Immediately return the tubes to the ice bath for at least 1 minute.
14. Sterilely add 800 µl of LB broth to each tube. Gently mix.
15. Allow cells to recover by incubating all three tubes at 37°C in a water bath for 30 min.
16. Review technique for making spread plates.
17. Following recovery, make spread plates for each culture dish by adding 100 µl of the appropriate
cells with a fresh sterile tip, and immediately spread with disinfected spreader. On the two plates that
require X-Gal and IPTG, add 25 µl and 10 µl each into your 100 µl puddle of cells and spread
normally.
18. Be sure to re-flame spreader before returning it to lab bench.
19. Place in wire basket, and place in 37°C incubator. Incubate for 2 days.
20. Recover the plates the next period.
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CLONING AND GENE EXPRESSION
LB/amp+kan
Table 3. Marking and loading the spread plates
Volume from tube
pLIG
pKAN-GFP
100 µl cells
100 µl cells
pGEM
100 µl cells
100 µl cells
LB/kan
100 µl cells
25 µl X-Gal
10 µl IPTG
100 µl cells
100 µl cells
100 µl cells
25 µl X-Gal
10 µl IPTG
100 µl cells
LB
100 µl cells
100 µl cells
100 µl cells
LB/amp
DAY 3: PLATE ANALYSIS AND PREPARATION OF
OVERNIGHT CULTURES
1. Count the number of colonies on each plate and record in the Table 4 below. If the colonies are too
numerous to count, record "lawn." Use the UV lamp to determine which colonies are green.
2. On your pLIG – LB/amp plate, identify a green, white, and blue colony.
3. Obtain 3 test tubes with 1 ml of sterile LB/amp broth. Label each with your team name and color of
colony.
4. With the inoculating loop, transfer each colony to the appropriate tubes (don't forget to flame the loop
for a sterile transfer). You do not have to scrape up the entire colony, a mere touch with your loop
will transfer thousands to millions of cells!
5. Make sure the tubes are appropriately labeled and place in the rack in the 37 oC incubator.
6. Your lab technician will move these tubes to 4 degrees C after 24 hours growth. The day before your
next lab period, the technician will add 4 ml fresh LB/amp broth and re-grow your cultures so that
they are ready for your miniprep and cell lysis.
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CLONING AND GENE EXPRESSION
Table 4. Colony counts in each plate
Color
LB/amp+kan
pLIG
pKAN-GFP
pGEM
Green
White
Green
LB/amp
White
Blue
LB/kan
LB
Green
White
Green
White
DAY 4:
MINIPREP
MATERIALS
3 overnight cultures made from colonies growing on the pLIG - LB/amp plate
GTE
SDS/NaOH
KOAc
Isopropanol
100% Ethanol
TE
Micropipets and tips
PROCEDURE
1. Disinfect work area.
2. Label three microfuge tubes to indicate your team name and original colony color.
3. Obtain your three overnight cultures and shake the tubes to resuspend cells.
4. Transfer 1.5 ml of culture into the correct microfuge tubes.
5. Centrifuge tubes for 1 minute.
6. Pour off supernatant into biohazard waste. Make sure the pellet remains in the tube.
7. Thoroughly drain the tubes by gently tapping on clean paper towel (be sure to discard towel in
biohazard container).
8. Add 100 µl of GTE solution to each tube. Resuspend pellets by pipetting solution in and out several
times. Hold tubes up to light to check that suspension is homogeneous and that no visible clumps of
cells remain.
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CLONING AND GENE EXPRESSION
9. Add 200 µl of SDS/NaOH solution to each tube. Close caps and mix by rapidly inverting tubes five
times.
10. Stand tubes on ice for 5 minutes. Suspensions should become relatively clear as the cells lyse.
11. Add 150 µl of ice-cold KOAc solution to each tube. Close caps, and mix solutions by rapidly
inverting tubes 5 times. A white precipitate will immediately appear.
12. Stand tubes on ice for 5 minutes.
13. Centrifuge tubes for 5 minutes to pellet the precipitate.
14. Transfer 400 µl of supernatant from each tube into a clean microfuge tubes, labeled appropriately.
(Save the supernatants)
15. Add 400 µl of isopropanol to each tube of supernatant.
16. Close caps and mix vigorously by rapid inversion five times.
17. Stand at room temperature for exactly 2 minutes. Isopropanol preferentially precipitates nucleic
acids, but with longer exposure proteins also begin to precipitate.
18. Centrifuge tubes for 5 minutes to pellet the precipitate. Hinge of microfuge tube up so you can tell
where to expect the nucleic acid residue.
19. Pour off supernatant from both tubes. Be careful not to disturb nucleic acid pellets. (Save the pellets)
20. Invert tubes over a paper towel and tap gently to drain thoroughly.
21. Rinse the pellets by adding 200 µl of 100% ethanol to each tube. Flick the tubes several times.
22. Centrifuge tubes for 2 minutes.
23. Pour off supernatant from both tubes. Save the pellets. Invert tubes over paper towel and drain
thoroughly.
24. Close caps and pulse tubes in centrifuge. Carefully remove any remaining ethanol with 2-20 µl
pipette.
25. Leave caps open and allow pellets to dry at room temperature for 10 minutes. All ethanol must be
evaporated before proceeding.
26. Add 15 µl of TE buffer to each tube. Resuspend pellets by smashing with the pipette tip and pipetting
in and out. Focus your efforts on the area where the pellet should have formed during centrifugation
(beneath the cap hinge). Check that all DNA is dissolved and that no particles remain in tip or on the
side of tube.
27. Keep the 3 DNA solutions separate. Do not pool into one tube.
28. Freeze the DNA/TE solutions at -20°C until you are ready to proceed with the restriction analysis of
the plasmid DNA.
LYSIS FOR CHROMATOGRAPHY
MATERIALS
Microtubes
Pipette
Microtube rack
Marker
Beaker of water for rinsing pipettes
TE solution
Lysozyme (rehydrated)
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CLONING AND GENE EXPRESSION
PROCEDURE
1. Transfer green culture into a microfuge tube and spin for 5 minutes in the centrifuge at maximum
speed. Be sure to balance the tubes in the machine.
2. Pour off the liquid supernatant above the pellet. After the supernatant has been discarded, there
should be a large bacterial pellet remaining in the tube. You may observe the pellet under UV light to
check GFP abundance.
3. Add 250 µl of TE Solution to each tube. Resuspend the bacterial pellet thoroughly by rapidly
pipetting up and down several times with the pipette.
4. Using a new blue pipette tip, add 1 drop of lysozyme to the resuspended bacterial pellet. Cap and mix
the contents by flicking the tube with your index finger. The lysozyme will start digesting the
bacterial cell wall. Observe the tube under the UV light. Place the microtube in the freezer until the
next laboratory period. The freezing will cause the bacteria to explode and rupture completely.
DAY 5
RESTRICTION ANALYSIS AND GEL ELECTROPHORESIS
MATERIALS
Miniprep DNA from previous lab (“Green, White, Blue”)
0.025 µg/µl λ marker (precut with HindIII)
0.075 µg/µl pKAN-GFP
0.075 µg/µl pGEM
BamHI/HindIII
5X restriction buffer/RNAase
DI water
Loading dye
0.8% agarose
1X Tris/Borate/EDTA (TBE) buffer
Micropipets and tips
Microfuge tubes
PROCEDURE
Tube
Green
White
Blue
pKAN-GFP
pGEM
λ marker
Table 5. Preparing tubes for restriction digest
DNA
Buffer/RNAase
BamHI/
HindIII
5 µl
3 µl
2 µl
5 µl
3 µl
2 µl
5 µl
3 µl
2 µl
5 µl
3 µl
2 µl
5 µl
3 µl
2 µl
10 µl supplied λ DNA cut with HindIII + 2 µl loading dye
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CLONING AND GENE EXPRESSION
1. Disinfect workspace.
2. Label 6 tubes according to the first column in the table below.
3. Use Table 5 as a guide to add the proper reagents to each tube. Read down each column, adding the
reagent to all appropriate tubes. Use a fresh tip for each reagent. Your lambda marker is already
prepared for you. All you need to do is load 12 µl on the gel.
4. When all tubes have their proper reagents, mix the tubes and pulse them in the microfuge.
5. Incubate at 37°C for 20 minutes.
6. Cast your agarose gel and prepare the gel box during the incubation
7. After removing tubes from the incubator, add 2 µl of loading dye to each tube.
8. Pulse in centrifuge to mix.
9. Load 12 µl of each tube into appropriate wells in gel in the same order they are in column 1 of the
table above.
10. Close gel box and connect to power supply. Electrophorese at about 100 volts until the smallest dye
marker has migrated about 3/4 of the way across gel.
11. Turn off power and remove gel from box. Take the gel to the lab technician for staining with
Ethidium Bromide.
12. The technician will photograph your gel.
Observe your data in table 4 and the photograph of your gel. The following are rules that are
required for a functional plasmid.
1. Every replicating plasmid must have an origin of replication. Recombinant plasmids with more than
one origin also replicate normally (however, only one origin is active).
2. Each adjacent restriction fragment can only ligate at a like restriction site, BamHI to BamHI and
HindIII to HindIII.
3. Repeated copies of a restriction fragment cannot exist adjacent to one another; that is they must
alternate with other fragments. Adjacent duplicate fragments form "inverted repeats" in which the
sequences, one on either side of the restriction site, are complementary along the entire length of the
duplicated fragment. Molecules with such inverted repeats cannot replicate properly. As the plasmid
opens up to allow access to DNA polymerase, the single strand regions on either side of the
restriction site base pair to one another to form a large "hairpin loop," which fouls replication.
CHROMATOGRAPHY
MATERIALS
Microtubes
Beaker of water for rinsing pipettes
HIC chromatography column
Column end cap
Waste beaker or tube
Binding Buffer
Equilibration Buffer
Collection tubes
Wash Buffer
Equilibration Buffer
TE Buffer
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CLONING AND GENE EXPRESSION
UV light
PROCEDURE
1. Remove your microtube from the freezer and thaw it out using hand warmth. Vortex vigorously to
resuspend. Place the tube in the centrifuge and pellet the insoluble bacterial debris by spinning for 10
minutes at maximum speed. Label a new microtube with your team’s initials.
2. While you are waiting for the centrifuge, prepare the chromatography column. Before performing the
chromatography, shake the column vigorously to resuspend the beads. Then shake the column down
one final time, like a thermometer, to bring the beads to the bottom. Tapping the column on the tabletop will also help settle the beads at the bottom. Remove the top cap and snap off the tab bottom of
the chromatography column. Allow all of the liquid buffer to drain from the column (this will take
~3–5 minutes).
3. Prepare the column by adding 2 ml of Equilibration Buffer to the top of the column, 1 ml at a time
using a well rinsed pipette. Drain the buffer from the column until it reaches the 1 ml mark which is
just above the top of the white column bed. Cap the top and bottom of the column.
4. After the 10 minute centrifugation from step 1, immediately remove the microtube from the
centrifuge. Examine the tube with the UV light. The bacterial debris should be visible as a pellet at
the bottom of the tube. Note the color of the pellet and the supernatant. Using a new pipette, transfer
250 µl of the supernatant into a new microtube. Again, rinse the pipette well for the rest of the steps
of this lab period.
5. Using the well-rinsed pipette, transfer 250 µl of Binding Buffer to the microtube containing the
supernatant.
6. Obtain 3 collection tubes and label them 1, 2, and 3. Place the tubes in a rack. Remove the cap from
the top and bottom of the column and let it drain completely into a liquid waste container. When the
last of the buffer has reached the surface of the HIC column bed, gently place the column on
collection tube 1. Do not force the column tightly into the collection tubes—the column will not drip.
7. Using a new pipette, carefully load 250 µl of the supernatant (in Binding Buffer) onto the top of the
column directly on the beads. Examine the column using the UV light. Note your observations in the
data table. Let the entire volume of supernatant flow into tube 1.
8. Transfer the column to collection tube 2. Using the rinsed pipette and the same loading technique
described above, add 250 µl of Wash Buffer and let the entire volume flow into the column. As you
wait, predict the results you might see with this buffer. Examine the column using the UV light and
list your results in the data table.
9. Transfer the column to tube 3. Using the rinsed pipette, add 750 µl of TE buffer (Elution Buffer) and
let the entire volume flow into the column. Again, make a prediction and then examine the column
using the UV light. List the results in the data table.
10. Examine all of the collection tubes using the UV lamp and note any differences in color between the
tubes.
Chromatography Data Table
Observations (color)
Tube 1
Tube 2
Tube 3
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CLONING AND GENE EXPRESSION
QUESTIONS FOR YOUR REPORT
1. Present an abstract of your work. Your abstract should include a brief description (1/2 page)
of 1) what you set out to learn, 2) what procedures you performed, and 3) a summary of your
data or discoveries.
2. Calculate the actual sizes of fragments generated from each of the restriction digests from
Day 1. These are your pKAN-GFP and pGEM plasmids cut with PstI/SalI and PstI/XhoI,
respectively.
3. Explain what each of the following plasmid parts does: Ori, AmpR, KanR, Gfp, LacZ.
4. Present your colony count data from table 4.
5. Explain what you expected to see on each of the LB plates in table 4 and what the purpose of
each plate was.
6. Explain what you expected to see on each of the LB/amp plates in table 4 and what the
purpose of each plate was. What is the significance of green, white, and blue colonies?
7. Explain what you expected to see on each of the LB/kan plates in table 4 and what the
purpose of each plate was. What is the significance of green and white colonies?
8. Explain what you expected to see on each of the LB/amp+kan plates in table 4 and what the
purpose of each plate was. What is the significance of green and white colonies?
9. Did you see any differences with what you expected in questions 5-8? If so, explain these
differences.
10. What would be an explanation for finding only white colonies on the pGEM-LB/amp plate?
11. Present your gel picture with all lanes properly labeled. Explain any irregularities or
unexpected results.
12. Present your standard curve generated using the λ marker.
13. Present your calculated estimations of fragment sizes in each lane on your gel. Include a table
with measured distances and calculated fragment sizes.
14. Based on your results, construct a map of each of the plasmid you created (green, white,
blue). Use actual fragment sizes/cut sites, not estimated.
15. Why is it impossible to have created a plasmid with an odd number of fragments?
16. Supposed you found a green colony on your pLIG-LB/amp+kan plate. Draw a map of a
possible plasmid this could be.
17. Explain the basic principle of HIC.
18. In preparing cells for chromatography, explain the importance of adding lysozyme and the
freezing step.
19. Present your data in the Chromatography Data Table. Explain why each tube was a certain
color in terms of what is happening at each step.
20. Describe how a researcher could apply the GFP protein in his/her research.
13