PROTOCOLS
Note: all protocols adapted from Edvotek kit 301 “Construction and Cloning of
Recombinant DNA”
Module I – Ligation of Vector to the Kanr Gene
1. Label and initial three 1.5ml microtest tubes 1-3:
“1”
“2”
“3”
stock ligation reaction mixture
ligation control
ligation reaction
2. Add the ligation reaction components to tube “1” in the following order:
A
B
→
→
40µl qualified water
20µl vector and kanr fragments
_____________________________
60µl Total volume
Mix by tapping or briefly vortexing.
3. With a fresh tip:




Remove 20µl of the ligation reaction from “1” and transfer to tube “2”.
Close tube “1”.
Add 5µl of 10x gel loading buffer to tube “2”.
Mix.
Set the tube aside – it is the control sample.
4. Vortex the T4 DNA Ligase Reaction Tube (C) and tap on lab bench to collect T4
ligase pellet at bottom of tube.
5. With a fresh tip, add all of the remaining stock ligation reaction mixture from tube
1 to the T4 DNA Ligase Reaction Tube. Re-label this tube 1 and incubate for 5
minutes at room temperature.
6. Carefully stir the mixture with a pipet tip and gently pipet the solution in tube 1
up and down to mix the DNA and ligase, briefly pulse in a microcentrifuge to
collect all of the solution at the bottom of the tube.
7. Incubate at room temperature (approximately 22ºC) for 1 hour or in a 16 ºC cool
waterbath for 30 minutes. Mix the tube by tapping or vortexing periodically
throughout the incubation period.
8. With a fresh tip:



Remove 20µl of the ligation reaction from “1” and transfer to tube “3”.
Close tube “1”.
Add 5µl of 10x gel loading buffer to tube “3”.
Mix
9. Tubes “2” and “3” will be electrophoresed and can be stored frozen until needed.
10. Continue with the experiment, or freeze the remainder of the ligation reaction
(tube 1) until needed for Transformation in Module II.
Preparing the Gel Bed
1. Close off the open ends of a clean and dry gel bed (casting tray) by using dams or
tape.
A. Using dams:

Place a black dam on each end of the gel casting tray in the
electrophoresis chamber. Make sure the dam is inserted
securely.
B. Taping with labeling or masking tape:


With ¾ inch wide tape, extend the tape over the sides and
bottom edge of the bed.
Fold the extended edges of the tape back onto the sides and
bottom. Press contact points firmly to form a good seal.
2. Place one well-formed template (comb) in the first set of notches nearest the end
of the gel bed and another comb in the middle set of notches. Make sure the
combs sit firmly and evenly across the bed. For convenience, place the comb
nearest the black electrode.
Casting the Gel
This experiment requires a 0.8% gel.
3. Use a 500-1000 ml flask to prepare the 500 ml of diluted gel running buffer.
NOTE: the gel running buffer concentrate is 50X.
AMOUNT OF 50X BUFFER =________
AMOUNT OF WATER =________
4. Transfer 100 ml of the freshly prepared 1X gel running buffer to a 250-300 ml
flask.
5. Add the required amount of agarose powder. Swirl to disperse clumps.
AMOUNT OF AGAROSE POWDER=______
AMOUNT OF BUFER=________
6.
With a marking pen, indicate the level of the solution volume on the outside of
the flask.
a. Heat the mixture to dissolve the agarose powder. The final solution should
be clear (like water) without any undissolved particles. It is a good idea to
set up a lukewarm water bath in which you will swirl the hot agarose
solution to accelerate cooling after the powder is dissolved.
A. Microwave method:
 Cover flask with plastic wrap to minimize evaporation.
 Heat the mixture on High for 1 minute.
 Swirl the mixture and heat on High in bursts of 25 seconds
until all the agarose is completely dissolved.
CAUTION!!! Be extremely careful when swirling the hot
solution; the extra kinetic energy you put in may cause the
superheated mixture to boil over. This is why you use a flask
that is 3X larger than the volume of the solution you are
making. Additionally, hold the flask with hot pads, hot gloves,
or paper towel collars.
B. Hot plate or burner method:
 Cover the flask with foil to prevent excess evaporation.
 Heat the mixture to boiling over a burner with occasional
swirling. Boil until all the agarose is completely dissolved.
7. Cool the agarose solution to ~55ºC with careful swirling in the water bath (if
available) to promote even dissipation of heat. If detectable evaporation has
occurred, add distilled water to bring the solution up to the original volume as
marked on the flask in step 6.
After the gel has cooled to 55ºC:
If using dams, go to step 9. If using tape, continue with step 8.
8. Seal the interface of the gel bed and tape to prevent the agarose solution from
leaking.
 Use a transfer pipet to deposit a small amount of cooled
agarose to both inside ends of the bed.
 Wait approximately 1 minute for the agarose to solidify.
9. Pour the cooled agarose solution into the bed in the electrophoresis chamber.
Make sure the apparatus is on a level surface.
10. Allow the gel to completely solidify. It will become firm and cool to the touch
after approximately 20 minutes.
Preparing the Gel for Electrophoresis
11. If using tape, carefully remove it from the casting tray after the gel is completely
solidified. If using the dams, pipette or pour a small amount of running buffer
around the comb to act as a lubricant for its subsequent removal,
12. Remove the comb by slowly pulling it straight up. Do this carefully and evenly to
prevent tearing the sample wells. Sometimes a slight rocking motion will facilitate
this process.
13. Remove the dams.
14. Fill the electrophoresis apparatus chamber with the diluted buffer so that the
liquid is ~1-3 mm above the highest point of the gel (near the wells).
15. Make sure the gel is completely covered with buffer. The agarose gel is
sometimes called a “submarine gel” because it is submerged under buffer for
sample loading and electrophoretic separation.
16. Each group should load 25µl of tubes “2” and “3” into adjacent sample wells.
Also load 5 µl of the 1kb Ladder marker in a 20µl final volume. Unless necessary,
use the inner wells as opposed to those on the edges. The bands tend to “smile”
less in the interior lanes (ex, lanes 2-7 in an 8-well gel).
17. Electrophorese samples until the bromophenol blue tracking dye has migrated 3
to 5 centimeters.
18. After electrophoresis is completed, stain the gel with ethidium bromide. The
solution you will be using will be made of 100 µl of 1 mg/ml EtBr in 300 ml H2O.
FINAL [EtBr]= ______
19. Stain the gel for ~10m, then transfer the gel to a short wave (300 nm) ultraviolet
transilluminator for viewing the DNA. Be sure to wear UV protective goggles.
Take a picture of the freshly stained gel.
20. Move the gel to a dish if distilled water for destaining. Leave 20m to overnight to
destain. If destaining overnight, perform the procedure in the cold room. Compare
the destained gel to the freshly stained one.
Module II – Transformation and Selection
1. Label the microcentrifuge tubes (1.5ml) “Ligation 1”, “Ligation 2”, and
“Control.” Or simply number and have the key available in your notebook.
2. With a sterile pipette, add 0.25ml ice cold 0.05M CaCl2 into each of the tubes and
place on ice.
3. With a sterile loop, transfer 1 single, well isolated colony from plate labeled
“HB101” to the tube “Ligation 1.” Twist the loop vigorously between your
fingers to dislodge the cells.
4. Vortex the cells to mix and fully suspend the cells in the CaCl2. Place on ice.
5. With a new loop, repeat the procedure with the “Control” tube.
6. With a new loop, repeat the procedure with “Ligation 2”, except transfer two
colonies to the tube.
7. Dilute the DNA from the ligation reaction by mixing 5µl of DNA from tube 1
(Module I) in 45µl qualified water (A). Vortex or tap tube with finger. Diluting
the DNA helps to minimize the carryover of excess salts from the ligation
reaction.
8. Add 10µl of the diluted ligation reaction DNA to the tube labeled “Ligation 1”
and 10µl of diluted ligation reaction DNA to the tube labeled “Ligation 2.”
Vortex or tap tubes with finger.
9. Add 5µl of supercoiled control DNA (5 ng/µl) for transformation (E) directly to
the tube labeled “Control.” Vortex or tap tube with finger.
10. Place all tubes on ice for 15 minutes.
11. Place tubes in 42ºC waterbath for 90 seconds.
12. Immediately, return tubes to ice for 2 minutes.
13. Aseptically pipet 0.25ml of recovery broth into each tube of cells.
14. Incubate cells for 30 minutes in 37ºC waterbath.
15. Place tubes in a microcentrifuge and spin for 5 minutes to pellet the cells.
16. Remove and discard 0.4ml of supernatant from each tube and resuspend cell
pellet in remaining 0.1ml liquid.
17. Label three plates, “Control”, “Ligation 1”, and “Ligation 2” and pipet 0.1ml of
the recovered transformed cells to the corresponding plates.
18. Using a sterile loop for each plate, spread the cells evenly and thoroughly over the
entire surface. Turn the plate ¼ turn and thoroughly spread again.
19. Incubate plates inverted in 37ºC oven overnight.
20. Estimate the number of transformants on both plates. Keep track of the counted
colonies by putting a dot over them on the outside of the plate with a lab marker.
21. Calculate the transformation efficiencies. The final recovery volume of the cells
was 0.5ml, the plated volume is 0.1 ml. and the amount of DNA used in the
control reaction is 25 ng. Explain the results.
Module III – Picking and Growth of Kan r Transformants
1. Obtain the tube of liquid kanamycin media and put your initials or lab group
number on it.
2. Using a sterile inoculating loop or needle, pick a SINGLE, well isolated colony
from your kanamycin agar plate, labeled “KanLigation.”
3. Inoculate the media. Tightly cap the tube.
4. Incubate the tubes at 37ºC, with shaking (400 rpm) overnight (12-15 hours).
Module IV – Extraction of Recombinant Plasmids from Kan r Transformants
1. Distribute all 10 ml of saturated the cell culture to 1.5 ml µfuge tubes.
2. Spin @ top speed in a µfuge for 2m.
3. Discard super, tap on paper towels to get rid of any residual liquid.
4. Resuspend the entire sample in 100 µl TEG (Tris-EDTA-Glucose) buffer. After
resuspending, you should have all 10 mls of the original overnight culture
resuspended in 100 µl TGE. Mix by pipetting (as you combine portions) and
vortexing.
5. Add 0.2ml of fresh NaOH/SDS cell lysis solution. Gently mix by inverting and
tapping.
6. Incubate on ice for 5 minutes.
7. Add 0.15ml of potassium acetate solution. Gently mix by inverting and tapping.
8. Incubate on ice for 5 minutes.
9. Centrifuge in a microcentrifuge (14,000 rpm) for 5 minutes. Make sure the
centrifuge is balanced.
7. Remove 0.4ml of the supernatant to a fresh 1.5ml µfuge tube. Try to avoid
drawing in the white precipitate. Set the old tube aside. Initial or put your lab
group number on the tube.
8. Thoroughly mix the deproteinization matrix (N) and quickly add 0.4ml to your
sample.
9. Mix the sample with matrix thoroughly by vortexing and inverting for 3 minutes.
10. Centrifuge 5 minutes in a microcentrifuge. Make sure the centrifuge is balanced.
11. Transfer 0.4ml of the aqueous phase (upper) to a fresh 1.5ml µfuge tube. Initial or
put your lab group number on the tube. Discard the old tube.
12. Add 0.8ml of ice-cold 95-100% ethanol. Mix well by inversion.
13. Incubate the tube on ice for 20 minutes.
14. Place your tube (with balance) in the microcentrifuge so that the tab which
connects the lid to the tube is facing the outside of the rotor. Centrifuge for 10
minutes at 14,000 rpm (top speed).
15. Remove and discard all the supernatant with a Pasteur pipette or automatic
pipette. Be careful not to dislodge the pellet which will be on the lower wall of the
tube or at the bottom. Do not touch the inner wall of the tube that was facing the
outside of the rotor.
16. Carefully add 0.25ml of ice cold 80% ethanol. Keep a close eye on the pellet
while doing this since it may dislodge at this step. Vortex, spin.
17. Immediately but carefully remove all the ethanol with a pipette, take care not to
aspirate the pellet. Alternatively, carefully pour out the ethanol. This step rinses
the tube of residual salts.
18. Air dry the tube 20 minutes or place under vacuum for 5 minutes.
19. Add 50µl TER (Tris-EDTA-RNase) buffer. Resuspend by thoroughly vortexing.
Module V – Restriction Enzyme Analysis
1. Make a reaction cocktail in a 1.5ml microtest tube and mix.
150µl qualified water (A)
25µl restriction reaction buffer (O)
35µl resuspended recombinant plasmid
2. Label four (4) 1.5ml microtest tubes 3-6. (Tubes 1 and 2 are standard DNA
fragments and supercoiled vector, respectively.)
3. Transfer 40µl of the cocktail to each tube.
4. Add 10µl of qualified water (A) to tube 3.
5. Add 5µl of qualified water (A) to tubes 4 and 5.
6. Add 5 µl (10-15 units) of diluted EcoRI endonuclease (S) to tube 4. Tap or
briefly vortex to mix.
7. Add 5 µl (10-15 units) of diluted Pvu II endonuclease (T) to tube 5. Cap. Mix.
8.
Add 5 µl of diluted Pvu II endonuclease (T) to tube 6. Then with a fresh pipet tip,
add 5 µl (10-15 units) of diluted Cla I endonuclease (U) to tube 6. Cap. Mix.
9. Incubate tubes 3 to 6 at 37ºC for 1 hour.
10. After the incubation, add 5µl of 10x gel loading solution to reaction tubes 3-6.
Mix.
11. Prepare agarose gel and apply samples to gel analysis as described in the next
section.
Separation of Restriction Enzyme Reactions by Electrophoresis
Prepare agarose gels according to instructions beginning on page 15.
Have a waterbath or beaker of water warmed to 65ºC for heating the tubes containing
DNA fragments before gel loading. At 65ºC, nonspecific aggregation due to sticky ends
generated by restriction enzyme digestions will melt. This will result in sharp individual
DNA bands upon separation by agarose gel electrophoresis.
Loading DNA Samples
1. Heat the standard DNA fragments (Q) for two minutes at 65ºC. Allow the
samples to cool for a few minutes.
2. Load samples in consecutive order in the wells.
Lane 1: Load 25µl Standard DNA Fragments (Q)
Lane 2: Load 25µl supercoiled (nonrecombinant) vector (R)
Lane 3: Load 25µl restriction enzyme control (recombinant plasmid)
Lane 4: Load 25µl Eco RI digest
Lane 5: Load 25µl Pvu II digest
Lane 6: Load 25µl of Pvu II/ Cla I codigest
Running the Gel
3. After the samples are loaded, carefully snap the cover down onto the electrode
terminals.
Make sure that the negative and positive indicators on the cover and
apparatus chamber are properly oriented.
4. Insert the plug of the black wire into the black input of the power source (negative
input). Insert the plug of the red wire into the red input of the power source
(positive input).
5. Set the power source at the required voltage and run the elctrophoresis for the
length of time as determined by your instructor. When current is flowing properly,
you should see bubbles forming on the electrodes.
6. Allow the tracking dye to migrate 3.5 to 4 centimeters from the wells for adequate
separation of the DNA bands.
7. After the electrophoresis is completed, turn off the power, unplug the power
source, disconnect the leads and remove the cover.
8. Stain gels as directed by Dr. Davis.
Size Determination of DNA Restriction Fragments
This is the first step for mapping DNA restriction sites, which is to determine the size of
the “unknown” DNA fragments generated after electrophoresis. The assignment of sizes
for DNA fragments separated by agarose gel electrophoresis can have ± 10% margin of
error. The sizes of the “unknowns” will be extrapolated by their migration distances
relative to the Standard DNA Fragments (Sample A), for which the size of each fragment
is known.
1. Measure and record the distance traveled in the agarose gel by each Standard
DNA fragment (except the largest 23,130 bp fragment, which will not fit in a
straight line in step 4).
In each case, measure from the lower edge of the sample well to the lower end of
each band. Record the distance traveled in centimeters (to the nearest millimeter).
2. Label the semi-log graph paper:
A. Label the non-logarithmic horizontal x-axis “Migration
Distance” in centimeters at equal intervals.
B. Label the logarithmic vertical y-axis “ Log base pairs”. Choose
your scales so that the data points are well spread out. Assume
the first cycle on the y-axis represents 100-1,000 base pairs and
the second cycle represents 1,000-10,000 base pairs.
3. For each Standard DNA fragment, plot the measured migration distance on the xaxis versus its size in base pairs, on the y-axis.
4. Draw the best average straight line through all the points.The line should have
approximately equal numbers of points scattered on each side of the line.
5. Measure the migration distance of each of the 3, 4, 5 and 6 fragments from
samples.