VGEC: Clone that Gene!
Gel electrophoresis from
recombinant clones
1) Gel electrophoresis of DNA from (recombinant) clones
Aim:
To load digested DNA fragments on gel to allow size separation.
Protocol:
The gels for this step have been prepared for you in advance. If you would like further
information on how to make and run gels A GENIE educational video will be shown,
explaining how to make and run a DNA agarose gel. This video is also viewable on-line on
the Virtual Genetics Education Centre a link to this video can be found in the information
on the provided USB memory drive.
Loading the gel
1. Add 6 µl of the blue gel-loading/STOP solution to each reaction mixture. Mix and
centrifuge briefly to collect the contents at the bottom of the tubes. Your reaction
mixtures now contain glycerol, a component of the loading dye, to make samples
sink to the bottom of the wells in the agarose gel.
2. Using the Gilson P20, load 15 µl of each reaction mixture (using a fresh tip each
time) into the appropriate wells of the gel, following the guide below (from left to
right). Also, load 12 µl of size marker DNA fragments to the wells at each end of
the row (lanes 1 and 16).
Lane 1.
Lane 2.
Lane 3.
Lane 4.
Lane 5.
Lane 6.
Lane 7.
Lane 8.
M.
1C
1B
1P
1E
1EP
1BP
1EB
Marker DNA
uncut plasmid 1
plasmid 1, BamHI
plasmid 1, PstI
plasmid 1, EcoRI
plasmid 1, EcoRI and PstI
plasmid 1, BamHI and PstI
plasmid 1, EcoRI and BamHI
Lane 9.
Lane 10.
Lane 11.
Lane 12.
Lane 13.
Lane 14.
Lane 15.
Lane 16.
2C
2B
2P
2E
2EP
2BP
2EB
M
uncut plasmid 2
plasmid 2, BamHI
plasmid 2, PstI
plasmid 2, EcoRI
plasmid 2, EcoRI and PstI
plasmid 2, BamHI and PstI
plasmid 2, EcoRI and BamHI
Marker DNA
3. When all the samples have been loaded, put the lid on the electrophoresis tank,
and connect the terminals to the power pack, as shown in the video. Make sure you
connect your apparatus correctly!
4. Set to 120 volts, and allow electrophoresis to continue for at least 60-90 min.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html
2) Transformation of E. coli
Aim:
To make E.coli take up the (recombinant) DNA molecules.
Protocol:
You are provided with:
Two DNA samples (plasmid 1 and plasmid 2), on ice
Three eppendorf tubes containing E.coli culture (25μl each tube) on ice
Sterile water, on ice
1 Luria agar plate containing tetracycline (labelled LT)
1 Luria agar plate containing ampicillin (labelled LA)
1 Luria agar plate containing kanamycin (labelled LK)
Luria broth (labelled LB)
Beaker of Chloros disinfectant. (WARNING, CORROSIVE)
1. Label the three eppendorf, tubes of bacteria 1, 2 and 3. Keep on ice!
2. To each of the labelled tubes, add the following using a P20 Gilson pipette:
Tube 1 5 μl plasmid 1
Tube 2 5 μl plasmid 2
Tube 3 5 μl sterile water
3. Dispense the sample INTO the cell suspension, not onto the side of the tube.
4. Use a fresh tip for each transfer; NEVER put a used tip into your DNA sample!!
5. Mix by flicking the tube and then shaking the contents to the bottom and leave on
ice for a further 5 minutes (n.b. usually this would be 30 minutes)
6. Heat shock the cells in all three tubes by incubating in the 42 oC water bath for 40
seconds.
7. Place tubes back on ice for 2 minutes.
8. Add 970 μl of Luria broth to each tube and incubate at 37 oC for 10 minutes (n.b.
usually this would be 1 hour).
9. Divide each of the Luria agar plates containing antibiotics (tetracycline, ampicillin
and kanamycin) into 3 sections and label the sections with the tube numbers (1, 2,
and 3). Inoculate the labelled sections of the three plates, by streaking out cultures
from the relevant tubes with a flame sterilised and cooled wire loop.
10. When the plates have dried they should be inverted and incubated at 37 oC.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html
3) Analysis of the phenotype of transformant clones
Aim:
To analyse the phenotypes of clones from transformation with plasmids 1 and 2.
Protocol:
You are provided with:
agar plates from a previous phenotype analysis experiment
Transformation efficiency
1. For each of the plasmids (1 and 2) look at the plates. Do you have colonies on the
plates where no DNA was added to the bacterial cells? Think why this is an
important control.
Phenotype test
2. For each of the plasmids (1 and 2) look at the transformation plates again. What
can you say about the phenotype of all transformant clones obtained with plasmid 1
and what can you say about the phenotype of all transformant clones obtained with
plasmid 1? So what can you say about plasmids 1 and 2 with respect to antibiotic
resistance genes?
3. Think of the implications of your observations.
Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html