Use Log/linear graph paper to calculate the size of each band inyour restriction digests (lanes 3-6) Look at the picture of the“1kb ladder” (Fig. I-18), and, using that as reference, assign asize for each of the bands in lane 2 (the sizes of the top andbottom bands are provided in the Table). Put these values into theTable provided. Using the ruler photographed with your gel, measurethe distance traveled for each fragment on the gel (all the bandsin the 1kb ladder AND all the bands in each of the other lanes ofthe gel) Enter the “distance traveled” from the “well” where youdeposited each sample for each band in each of the gel lanes (lanes#2, #3, #4, #5, #6) in the Table provided. As an example, uncutplasmid in lane #3 MAY have 3 different bands. You’ll give theirdistances traveled with the top band being the one closest to thegel origin. Your distances should be accurate to 0.1cm (1mm). Firstgenerate a STANDARD CURVE from the information you have for your“1kb ladder.” Note that for each band in this lane, you know BOTHthe size of the fragment AND the distance that the fragmenttraveled. Assign a “dot” on the log/linear graph for each fragmentin the “1kb ladder”, indicating distance traveled on the X-axis(horizontal axis = linear scale) and the size of the fragment onthe Y-axis (vertical axis = logarithmic scale). You should plot 20dots for the “1kb ladder”. When all dots are placed, draw astraight line from each dot to the next. You have now created yourSTANDARD CURVE (see an example in Fig. I-19). Now determine thesize of all the other fragments on your gel. You have alreadydetermined the distance traveled for each fragment. Use a pencil tofind the place on the X-axis corresponding to that distance. Draw astraight line up (vertical line) from that place on the X-axisuntil it intersects with the STANDARD CURVE. At that point, draw astraight, HORIZONTAL LINE to the left until you reach the Y-axis.The point of intersection on the Y-axis = size of the fragment.
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1Kb DNA Ladder ፐ ፐ 2,000 ← 1,650 ↑↑ ↑ up 12,000 ↑ ↑ ↑ ↑ 5,000 G- 1,000 850 650 500 400 300 200 100 1 Kb Plus DNA Ladder (Invitrogen) 0.7 µg/lane 0.9% agarose gel stained with ethidium bromide Structure of Fragments in 1-Kb Increments: 5'-GATCC-- G -CCTAG-5' (996 bp),
CM 11 17 BELL