Introduction to Sizing DNA

on an Agarose Gel

HC 70AL
Spring 2009
April 2, 2009
By Kristin Gill
How Do We Determine the Size of a PCR
Product?

Fractionate a PCR product along with a DNA

ladder by gel electrophoresis

Compare the migration distance of the PCR

fragment to that of the DNA ladder
 What Is a DNA Ladder?

A DNA solution composed of many DNA

fragments with different known lengths

A ladder is used as a reference to estimate the

size of an unknown DNA fragment
What Ladders Do We Use in This Laboratory?
1-kb ladder 100-bb ladder
(Invitrogen) (Invitrogen)

12,216 bp
2,072 bp
1,500 bp

4,072 bp
3,054 bp
QuickTime
600 bp and a
decompressor
2,036 bp
QuickTime and a
are needed to see this p
decompressor
are needed
1,636 bpto see this picture.
1,018 bp

506/517 bp
396 bp 100 bp
 How Can We Estimate the Size of the
Unknown DNA Fragment?

1. Create a standard curve of DNA ladder fragments

and their migration distance using either a
logarithmic graph or an Excel program

2. Estimate the size of the unknown DNA fragment

based on its migration distance relative to the
standard curve
 Creating a Standard Curve

Measure how far each band traveled using a metric ruler (cm)
Example:

DNA Ladder Fragment Distance Traveled (cm)

Size (bp)
1500 1.0
1200 1.34

1000 1.5
900 1.7
800 1.9
700 2.2
600 2.7
500 3.7
400 4.9
 Creating a Standard Curve
Plot the data points using an Excel program

1600

1400

1200

1000 Logarithmic
Trend line
800

600

400

200

0
0 2 4 6 8 10
 How Is the Size of the Unknown DNA
Fragment Determined?
Measure the distance traveled by the unknown
fragment
Interpolate the size based on the standard curve

1600

1400
Example: 2 cm
1200
Fragment size: 875 bp
1000
875
800

600

400

200

0
0 2 4 6 8 10
 Why Do You Need to Know the Size of the
PCR Product?
Sequencing!
The amount of DNA used in the sequencing
reaction is determined according to the size of
the fragment (in bp) recommended by Applied
Biosystems protocol
Size of PCR Product Amount of DNA Used in
(bp) Sequencing Reactions
100-200 1-3 ng
200-500 3-10 ng

500-1000 5-20 ng

1000-2000 10-40 ng

(Taken from Experiment 1: Intro. to General Molecular..Page 1.21)