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(Invitrogen)

QuickTime™ and a decompressorare needed to see this picture.600 bp

100 bp

1,500 bp2,072 bp

QuickTime™ and a decompressorare needed to see this picture.

12,216 bp

2,036 bp1,636 bp

3,054 bp

1,018 bp

506/517 bp396 bp

4,072 bp

100-bb ladder(Invitrogen)

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 Size (bp)

Distance Traveled (cm)

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

Distance Traveled vs. Fragment Size

0

200

400

600

800

1000

1200

1400

1600

0 2 4 6 8 10

Distance Traveled (cm)

Fragment Size (bp)

Logarithmic Trend line

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

Distance Traveled vs. Fragment Size

0

200

400

600

800

1000

1200

1400

1600

0 2 4 6 8 10

Distance Traveled (cm)

Fragment Size (bp)

875

Example: 2 cm

Fragment size: 875 bp

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 (bp)

Amount of DNA Used in 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)