Cloning Worksheet
Winter 2011
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
• 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.
Standard Semi-Log Plot
Molecular Weight (base pairs)
100,000
10000
1000
100
Distance Traveled (cm)
Standard Semi-Log Plot
Distance Traveled (cm)
1.8
Distances
1.9
measured
2.0
from gel
photograph
2.1
2.25
2.5
2.8
3.15
3.75
4.65
MW (bp)
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
Standard
sizes of the
“ruler”
fragments
100,000
Omit data that
doesn’t show a
linear relationship.
10000
X
XX
X
X
Use the best fit
line.
X
X
X
1000
X
X
100
1
2
3
4
5
100,000
Finding the size of an unknown fragment
10000
X
XX
X
X
MW is
~900 bp
X
X
X
1000
X
X
Fragment
at 3.8 cm
100
1
2
3
4
5
Before plotting the
standard curve, use
the Excel function
=LOG10(x)
to convert MW
numbers to log
values
Log Molecular Weight (bp)
Using Excel to
Estimate Fragment Size
4
3.5
3
2.5
Log MW
2
Linear (Log MW)
1.5
1
0.5
0
0
2
4
6
Distance Traveled (cm )
Trendline equation: 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