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) 600 bp 100 bp 1,500 bp 2,072 bp 12,216 bp 2,036 bp 1,636 bp 3,054 bp 1,018 bp 506/517 bp 396 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)

Creating a Standard Curve Plot the data points using an Excel program 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 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 ng ng ng ng (Taken from Experiment 1: Intro. to General Molecular…..Page 1.21)