cloning worksheet winter 2009
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Cloning Worksheet Winter 2009. Producing a Standard Curve to Determine DNA Fragment Sizes. Measuring Distance Traveled on Agarose Gel. Using a ruler and gel photograph, measure how far each DNA fragment traveled from its loading well - PowerPoint PPT PresentationTRANSCRIPT
Cloning WorksheetWinter 2009
Producing a Standard Curve to Determine DNA Fragment Sizes
Measuring Distance Traveledon Agarose Gel
• Using a ruler and gel photograph, measure how far each DNA fragment traveled from its loading well
• Choose a consistent starting point, either the middle or edge of the well
• Measure from the starting point to the middle of each DNA band in metric units
• Plot the log of the molecular weight (in base pairs) versus the distances traveled for the standard “ruler” fragments
• Put a best fit line through these points• Determine the sizes of the PCR products, plasmids and
inserts by interpolating from the standard curve of the ruler fragments
• You may use semi-log paper to plot the line by hand or complete the analysis in Excel
Measuring Distance Traveled
Measure from the
well to the middle of each band.
Plot the standard
curve with these values.
Measure the distance for
plasmids and fragments.
Use the standard
curve to find their sizes.
1000
100
10000
100,000Standard Semi-Log Plot
Distance Traveled (cm)
Mol
ecul
ar W
eigh
t (ba
se p
airs
)
Standard Semi-Log Plot
Distance Traveled (cm) MW (bp)
1.8 5000
1.9 4500
2.0 4000
2.1 3500
2.25 3000
2.5 2500
2.8 2000
3.15 1500
3.75 1000
4.65 500
Standard sizes of the
“ruler” fragments
Distances measured from gel
photograph
1000
100
10000
100,000
1 2 3 4 5
X
XX
XX
XX
XXX Use the best fit
line.
Omit data that doesn’t show a
linear relationship.
1000
100
10000
100,000
1 2 3 4 5
X
XX
XX
XX
XXX
Fragmentat 3.8 cm
MW is ~900 bp
Finding the size of an unknown fragment
Using Excel to Estimate Fragment Size
y = -0.3442x + 4.2808
Solve for y using x = 3.8: y=2.97, Raise 10 to the y power: 102.97 = 939 base pairs
0
0.5
1
1.5
2
2.5
3
3.5
4
0 2 4 6
Distance Traveled (cm)
Lo
g M
ole
cula
r W
eig
ht
(bp
)
Log MW
Linear (Log MW)