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11, April 2007 Biology 214 Restriction Enzyme Results and Discussion The agarose gel electrophoresis of lambda HindIII, EcoRI digested, PstI digested, and Lambda DNA showed that the fragments varied in size (Figure 1). Lambda DNA has the largest fragments which is obvious because they migrated the shortest distance. Fragments that are the same size will move together through the gel and form bands (Biology 214 Laboratory Manual). Figure 1. Agarose gel electrophoresis photograph. The wells, from top to bottom are the marker, Lambda DNA, PstI digested, EcoRI digested, and lambda HindIII. From the known number of base pairs in HindIII, estimations can be made about the other samples (Table 1).The farther the band migrates the fewer base pairs there are. To more accurately estimate the unknown DNA band sizes, a standard curve based upon the band sizes of the HindIII bands can be made (Figure 2). Table 1. An estimation of base pairs per band for Lambda DNA, PstI digested, and EcoRI digested based on the number of base pairs on HindIII DNA per millimeter. migrated. Lambda HindIII Distance Base Log Migrated Pairs bp 13 23130 4.4 15 9416 4.0 17 6657 3.8 19 4361 3.6 24 2322 3.4 26 2027 3.3 EcoRI Base Pairs 23130 8370 5271 3896 3103 PstI Base Pairs 9416 3896 2950 3230 2322 2027 Lambda DNA Distance Base Log Migrated Pairs bp 7 40289 4.6 11 30574 4.5 Band 1 2 3 4 5 6 Distance Migrated 13 16 18 20 21 Log bp 4.4 3.9 3.7 3.6 3.5 Distance Migrated 15 20 22 23 24 26 Log bp 3.97 3.6 3.5 3.5 3.37 3.31 After the standard curve is made the 5 Log base pairs size of the known fragments from the DNA standards can be used against the distance each DNA band moved through the gel, to generate a standard curve (Figure 3). Then it is possible to accurately estimate the number of base pairs for the unknown DNA samples (Table 2). 4 3 2 1 0 0 10 20 30 Distance Migrated (m m ) Figure 2. The distance migrated in millimeters of HindIII in log base pairs. 25000 20000 Base Pairs 15000 10000 5000 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Distance Migrated (mm) Figure 3. The distance in millimeters migrated by HindIII in base pairs. The equation y=-1261.2x + 31949 can be used to find the exact number of base pairs for the unknown DNA fragments. Table 2. An estimation of base pairs per band for Lambda DNA, PstI digested, and EcoRI digested based on the equation y=-1261.2x 31949 + from the standard curve of HindIII DNA. Band 1 2 3 4 5 6 Distance Migrated 13 15 17 19 24 26 HindIII Base Pairs 23130.0 9416.0 6657.0 4361.0 2322.0 2027.0 Log bp 4.4 4.0 3.8 3.6 3.4 3.3 Distance Migrated 13 16 18 20 21 EcoRI Base Pairs 15554 11770 9247.4 6725.0 5463.8 Log bp 4.2 4.1 4.0 3.8 3.7 Distance Migrated 15 20 22 23 24 26 PstI Base Pairs 13031.0 6725.0 4202.6 2941.4 1680.2 2027 Log bp 4.1 3.8 3.6 3.5 3.2 3.3 Lambda DNA Distance Base Log Migrated Pairs bp 7 23120.6 4.4 11 18075.8 4.3 The objectives of this lab were to cut lambda DNA into fragments using restriction enzymes, separate and sort DNA molecules according to their size, and to determine the size of each molecule separated by gel electrophoresis (Biology 214 Laboratory Manual). The HindIII lambda DNA was the known standard. It had between 23130 and 2027 base pairs. The EcoRI digested DNA also started at thirteen millimeters but was estimated to only have 15554 base pairs. The PstI digested DNA migrated the farthest and had a maximum of 13031 base pairs. The Lambda DNA had very large fragments; it migrated a maximum of eleven millimeters. At the beginning of the lab the EcoRI, PstI, and HindIII samples of DNA were all the same size. Then each sample was cut into pieces by the addition of three different restriction enzymes. The data shows that the three samples of DNA were all cut in different places, showing that each of the enzymes cut independently of each other. The PstI enzyme made the smallest fragments of DNA, which is why it traveled the farthest in the gel. The EcoRI made the largest fragments of the three enzymes, which lead to the least migration. This enzyme cuts the palindromic sequence guanine, adenine, adenine, thiamine, thiamine, cytosine (Biology 214 Laboratory Manual), so it can be inferred that this sequence was less abundant than the sequence the PstI enzyme corresponds to. There are some possible errors that could have affected the results. When originally measuring the picture of the gel electrophoresis, putting a ruler against the computer screen is not the most precise way to measure. It would be better to use a program on the computer that could measure the bands electronically. Literature Citied Biology 214 Laboratory Manual. 2007. Restriction Digestion and Analysis of Lambda DNA Kit, pp. 19-44. Case Western Reserve University, Cleveland, OH.

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