gel electrophoresis experiment fall 2007
DESCRIPTION
Gel Electrophoresis Experiment Fall 2007. A Timed Presentation - do not click the mouse. Approximate Run Time - 4 minutes 5 seconds. Comb produces 32 wells. Gel Apparatus. Gel is 22.5 x 22.5 cm in size. Restriction Enzyme Digest Oven. Temperature accuracy is good to + or - - PowerPoint PPT PresentationTRANSCRIPT
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.