1. Cloning Worksheet Winter 2011 Producing a Standard Curve to Determine DNA Fragment Sizes

2. Measuring Distance Traveled on 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

3. 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.

4. 1000 100 10000 100,000 Standard Semi-Log Plot Distance Traveled (cm) Molecular Weight (base pairs)

5. Standard Semi-Log Plot Distance Traveled (cm)MW (bp) 1.85000 1.94500 2.04000 2.13500 2.253000 2.52500 2.82000 3.151500 3.751000 4.65500 Standard sizes of the “ruler” fragments Distances measured from gel photograph

6. 1000 100 10000 100,000 1 2 345 X X X X X X X X X X Use the best fit line. Omit data that doesn’t show a linear relationship.

7. 1000 100 10000 100,000 1 2 345 X X X X X X X X X X Fragment at 3.8 cm MW is ~900 bp Finding the size of an unknown fragment

8. 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: 10 2.97 = 939 base pairs Before plotting the standard curve, use the Excel function =LOG10(x) to convert MW numbers to log values