A Timed Presentation - do not click the mouse. Approximate Run Time - 2 minutes 38 seconds

Comb produces 28 wells Gel is 22.5 x 22.5 cm in size

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 Bouble 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 °C. Each sample and the ladder was then loaded into each well as outlined in the following slides. pBabe/cpp bp BamHI EcoRI 900 bp 5100 bp pBabe plasmid donated by Dr. Tang

Note die markers

Note separation of die markers

Ladder Rows:

Ladder Rows:

2 To 7 Uncut Plasmid Rows

2 To 7 Uncut Plasmid Rows

Single Digest Rows 9 to 17 HindIII

Single Digest Rows 9 to 17 HindIII

Double Digest Rows 19 to 27

Double Digest Rows 19 to 27

2000 bp3000 bp4000 bp5000 bp8000 bp12000 bp 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.

2000 bp5000 bp6000 bp Single cut (BamHI) plasmid runs at the 6000 bp length.

2000 bp850 bp5000 bp12000 bp Double cut (BamHI; EcoRI) plasmid runs at just over the 5000 bp length and a second smaller piece at approximately 850 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.