Cloning Worksheet Winter 2011

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

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

5. Standard Semi-Log Plot Distances measured from gel photograph Standard sizes of the “ruler” fragments

6. X X X X X X X X X 100,000 Omit data that doesn’t show a linear relationship. 10000 Use the best fit line. 1000 X 100 1 3 4 5 2

7. 100,000 Finding the size of an unknown fragment 10000 X X MW is ~900 bp X X X X X X 1000 X X Fragmentat 3.8 cm 100 1 3 4 5 2

8. Using Excel to Estimate Fragment Size Before plotting the standard curve, use the Excel function =LOG10(x) to convert MW numbers to log values Trendline equation: y = -0.3442x + 4.2808 Solve for y using x = 3.8: y=2.97, Raise 10 to the y power: 102.97 = 939 base pairs