have plotted the known fragment sizes, a straight line is drawn, and that then enables you to determinethe size, in base-pairs, of the unknown fragment by comparing it to the migration distance of the standards. Lambda HindIIINote: Two smaller fragments of 564 and 125 bp are sometimes seen as faint bands at the bottom of the gel Standard Curve using Lambda HindIIINote: The graph shown above is an example; you will want to construct a similar curve using the data from your gel photographExample: An unknown fragment runs to 25mm. A line is drawn up to the straight line from the X-axis, and horizontally over to the Y-axis. The BP value read on the Y-axis is about 3300bp.You will now create a standard curve using your data: You have been provided with three-cycle semi-log graph paper. Fragment size, in bp, will be on the y-axis. Distance traveled through the gel, in mm, will be on the x-axis.
Electrophoresis data: (Week 2)Using the photograph of the gel, Measure the distance in mm that each fragment traveled from the well and record it in the table. Estimate the size, in base pairs, by comparing its position to the HindIII cut Marker DNA. Some lanes will have fewer or more than 6 fragments, but you do not need to record more than 6 bands.MarkerDNALambdaDNA uncutPst1 cut DNAEcoRI cut DNAHindIIIcut DNADistance(mm)Actual base PairsDistance(mm)Est. base pairsDistance(mm)Est. base pairsDistance(mm)Est. base pairsDistance(mm)Est. base pairsBand 123,130Band 29,416Band 36,557Band 44,361Band 52,322Band 62,027QUESTIONS:1)What is the source of the DNA being used in this exercise? 2)Why do bacteria make restriction enzymes ‘in nature’?3)Why is the restriction site for Eco RI, GAATTC, called a palindrome?4)What is a Sticky End? 5)If the line below represents a DNA double helix, stretched out, and the arrows indicate the sequence GAATTC, how many fragments would be produced if the DNA is cut by the enzyme EcoRI?6)Number each fragment7)Which fragment would be the largest?8)Which fragment would be the smallest?9)Which fragment would migrate the fastest on an agarose gel? The slowest?
10) The speed of the DNA fragment migration correlates directly with the _____ of the fragment. 11) What is agarose, and why is it used in this experiment?12) What are TWO purposes of the Loading Dye in this experiment?13) What is the purpose of the TBE buffer in this experiment?14) What is the purpose of the Gel Red in this experiment?
You've reached the end of your free preview.
Want to read all 8 pages?