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Cloning Worksheet Winter 2011: Producing A Standard Curve To Determine DNA Fragment Sizes

The document provides instructions for producing a standard curve to determine DNA fragment sizes using agarose gel electrophoresis. It describes measuring the distance traveled by DNA standards of known sizes on a gel and using those measurements to plot a standard curve of log molecular weight versus distance. This standard curve can then be used to interpolate the sizes of unknown DNA fragments by measuring their distance traveled on the gel.

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KAILEY
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30 views8 pages

Cloning Worksheet Winter 2011: Producing A Standard Curve To Determine DNA Fragment Sizes

The document provides instructions for producing a standard curve to determine DNA fragment sizes using agarose gel electrophoresis. It describes measuring the distance traveled by DNA standards of known sizes on a gel and using those measurements to plot a standard curve of log molecular weight versus distance. This standard curve can then be used to interpolate the sizes of unknown DNA fragments by measuring their distance traveled on the gel.

Uploaded by

KAILEY
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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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 Measure the
well to the distance for
middle of plasmids and
each band. fragments.
Plot the Use the
standard standard
curve with curve to find
these values. their sizes.
Standard Semi-Log Plot
100,000

Molecular Weight (base pairs)

10000

1000

100
Distance Traveled (cm)
Standard Semi-Log Plot
Distance Traveled (cm) MW (bp)
1.8 5000
Distances
1.9 4500 Standard
measured sizes of the
from gel 2.0 4000
ruler
photograph 2.1 3500 fragments
2.25 3000
2.5 2500
2.8 2000
3.15 1500
3.75 1000
4.65 500
100,000
Omit data that
doesnt show a
linear relationship.

10000
X Use the best fit
XX
X line.
X
X
X
X
1000 X

100
1 2 3 4 5
100,000

Finding the size of an unknown fragment

10000
X
XX
MW is X
X
~900 bp X
X
X
1000 X

Fragment
at 3.8 cm
100
1 2 3 4 5
Using Excel to
Estimate Fragment Size
Before plotting the
standard curve, use
the Excel function
=LOG10(x)
to convert MW
numbers to log
values

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

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