A Timed Presentation - do not click the mouse. Approximate Run Time – 4 minutes and 45 seconds

II. A 1% agarose solution was used to make the gel. Distilled water was heated till near boiling when 1 g/100 ml of water was added slowly to ensure no lumping of the agarose. The solution was stirred using a magnetic stirrer – while heating. III. The agarose solution was cooled to the touch before pouring into the mold. The agarose solution was poured carefully to ensure no air bubbles ended up in the mold and that the combs to produce the wells were well covered. I. The gel electrophoresis apparatus consists of a central plate (where the gel will sit) and two wells - one at each end. The mold for the agarose gel was constructed by applying masking tape at each end of the plate to seal the agarose from pouring into the wells. After the agarose hardened, the masking tape and well comb was removed.

Front View Expanded View of comb showing the 28 gel wells Electrode Connections+ve-ve

Back View Large end well connector to ensure fluid levels remain the same Tape used for pouring the agarose gel. Removed after gel hardens.

Comb produces 28 wells

Uncut:DNA18.75 μl 10 x Buffer15 μl Water μl 6 x Loading Buffer30 μl 30 μl/well – approximately 1.25 μg DNA/well Single cut:DNA37.5 μl 10 x Buffer22.5 μl BamHI15 μl Water150 μl 6 x Loading Buffer45 μl 30 μl/well – approximately 1.67 μg DNA/well Double cut:DNA37.5 μl 10 x Buffer22.5 μl BamHI11.25 μl EcoRI11.25 μ l Water142.5 μl 6 x Loading Buffer45 μl 30 μl/well – approximately 1.67 μg DNA/well Each sample of DNA was incubated for 1 hour at 37 ± 1-2°C (due to incubator). Each sample and the ladder was then loaded into each well as outlined in the following slides. Marker (ladder): 2 μl Water 8 μl 6 x Loading Buffer 30 μl

pBabe/cpp bp BamHI EcoRI 900 bp 5100 bp pBabe plasmid donated by Dr. Tang Associate Professor at McMaster University Hamilton, Ontario

Note separation of die markers

Note further separation of die markers

Uncut Plasmid Rows 2 to 7

Uncut Plasmid Rows 2 to 7

Single Digest Rows HindIII 9 to 17

Single Digest Rows HindIII 9 to 17

Double Digest Rows HindIII & EcoR1 19 to 25

Double Digest Rows HindIII & EcoR1 19 to 25

Uncut, circular plasmid runs as a smear slightly smaller than the size of the actual number of base pairs. The smear is seen mainly in the 3000 – 4000 bp length, but a small portion can be seen at 8000 – 9000 bp bp3000 bp4000 bp5000 bp 8000 bp 9000 bp

Double cut (BamHI; EcoRI) plasmid runs at just over the 5000 bp length and a second smaller piece at approximately 850 bp (*the smaller 850 bp piece ran off the end into the well due to a miscalculation on the run time for the gel. Single cut (BamHI) plasmid runs at the 6000 bp length bp2000 bp 3000 bp4000 bp 5000 bp 6000 bp 7000 bp

The SBI4U class of Hill Park Secondary School wish to thank Dr. D. Tang (McMaster University/St. Joseph’s Hospital) and his lab technician for donating the DNA, Restriction Enzymes and Ladder and for calculating the amounts of material required to complete this procedure successfully.