gel electrophoresis experiment fall 2007

A Timed Presentation - do not click the mouse.

Approximate Run Time - 4 minutes 5 seconds

Gel is 22.5 x 22.5 cm in size

Comb produces 32 wells

Uncut: DNA 18.75 μl10 x Buffer 15 μlWater 116.25 μl 6 x Loading Buffer 30 μl

30 μl/well – approximately 1.25 μg DNA/well

Single cut: DNA 37.5 μl10 x Buffer 22.5 μl

BamHI 15 μlWater 150 μl 6 x Loading Buffer 45 μl

30 μl/well – approximately 1.67 μg DNA/well

Bouble cut:DNA 37.5 μl10 x Buffer 22.5 μlBamHI 11.25 μlEcoRI 11.25 μ lWater 142.5 μl 6 x Loading Buffer 45 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/cpp326000 bp

BamHI

EcoRI

900 bp

5100 bp

pBabe plasmid donatedby Dr. Tang

LadderRows:

1

8

18

28

2

To

7

UncutPlasmid

Rows

SingleDigestRows

9

to

17

HindIII

DoubleDigestRows

19

to

27

2000 bp 3000 bp 4000 bp 5000 bp 8000 bp 12000 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 bp 5000 bp 6000 bp

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

2000 bp850 bp 5000 bp 12000 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 ofHill Park Secondary School

wish to thank

Dr. D. Tang

(McMaster University/St. Joseph’s Hospital)and his lab technician

for donating the DNA, Restriction Enzymesand Ladder and for calculating the amounts

of material required to complete this procedure successfully.