Cloning WorksheetWinter 2011

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

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

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)

Before plotting the standard curve, use the Excel function

=LOG10(x) to convert MW numbers to log

values