Introduction to Sizing DNA on an Agarose Gel

1
Introduction to Sizing DNA on an Agarose Gel

• HC 70AL
• Spring 2009
• April 2, 2009
• By Kristin Gill

2
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

3
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

4
What Ladders Do We Use in This Laboratory?

• 1-kb ladder
• (Invitrogen)

100-bb ladder (Invitrogen)
5
How Can We Estimate the Size of the Unknown DNA
Fragment?

• Create a standard curve of DNA ladder fragments
  and their migration distance using either a
  logarithmic graph or an Excel program
• Estimate the size of the unknown DNA fragment
  based on its migration distance relative to the
  standard curve

6
Creating a Standard Curve

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

7
Creating a Standard Curve

• Plot the data points using an Excel program

Logarithmic Trend line
8
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

Example 2 cm Fragment size 875 bp
9
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

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