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Experiment 2: Introduction to Recombinant DNA Methodologies...

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Experiment 2: Introduction to Recombinant DNA Methodologies

Part 1: Isolation of DNA from Spinach Leaf Part 2: Agarose Gel Electrophoresis Analysis of Spinach DNA Purification and Digests of Lambda Phage DNA

Name: Hannah Philip Lab Partner: Pearce Towindo Lab Room: 406 Lab Time & Day: Wednesday AM (Part 1 – January 23, 2019) (Part 2 – January 30, 2019)

Abstract The experiment of recombinant DNA methodologies was divided into two parts over the course of two weeks. The first part consisted of the isolation and extraction of DNA from spinach leaf that was done using a commercial DNA extraction kit and the purity of the DNA was evaluated using a spectrophotometer to record its absorbance at 260nm and 280nm, individually. The purity of the spinach leaf DNA was found to be 1.66, which is an impure DNA sample. The second part of the experiment consisted of digesting the restriction enzymes EcoRI and HindIII with Lambda DNA individually and was compared to the digestion of spinach leaf DNA, which was mixed with reaction buffer. A fourth tube was prepared and contained no restriction enzyme, which served as the control, also known as the uncut Lambda DNA. The products of digestion were further analyzed using agarose gel electrophoresis. The results of the electrophoresis show that restrictive enzymes digested with Lambda showed distinctive bands of fragments compared to that of the spinach leaf DNA in reaction buffer, which appeared as a smear on the gel image. The bands correspond to the exact fragments formed by each digestion of the restriction enzymes with the lambda DNA and the smear on the gel corresponds to the multiple tiny fragments formed by the spinach DNA digestion, which may also contain impurities.

TABLE 1. Concentration and Purity data of DNA from spinach leaf after isolation Percent Mass of DNA Purity of DNA

Absorbance

Concentration of

Coefficient of

Purified DNA (μg/mL) a in original leaf tissue

(A260/A280) c

Sample (%) b

pure DNA at 260 nm  cm) (mL / μg

0.020 a

12.54

0.001254

1.66

Concentration of DNA calculated using the Beer-Lambert Law (A= Elc) where E is

0.02cm-1 (μg/mL). b Mass percentage was calculated using 100mg of the original wet tissue of leaf. c Purity was calculated using the absorbance ratio between average absorbance values at 260nm and 280nm.

DNA Ladder

E

H

UC

RB

E

H

UC

RB

10kb 8kb 6kb 5kb 4kb 3kb

2kb 1kb 0.5kb

Figure 1. Gel Image of digested Lambda DNA and spinach leaf DNA. The gel consisted of 0.6% molten agarose (in TRIS acetate EDTA (TAE) buffer). The gel was loaded with 5μL of the DNA ladder, 15μL of each restriction digest product Lambda DNA (40μg/μL) with restriction enzymes EcoRI (E) and HindIII (H), and one with a restriction buffer into lanes E,H, and UC respectively. Finally, 25μL of the spinach leaf sample was loaded with a reaction buffer into the RB lane.

5. In order to evaluate DNA purity, the ratio of absorbance at 260nm and 280 was calculated and found to be 1.66, indicating the DNA had low purity1. Lower ratios indicate there are contaminants present in the DNA and according to literature values; relatively pure DNA will be more than 1.7 and ideally 1.81. The quality of DNA can also be evaluated by observing the bands present on the gel electrophoresis. A good quality DNA sample will yield a clear band and poor quality DNA samples may show up as a smear2. The DNA sample loaded showed a single smeared band, indicating that it was of a low quality with possibilities of the presence of contaminants in the DNA sample or the sample may have been too concentrated2. 71 f(x) = − 32.77 66 x + 58.56

Migration distance (mm)

61 56 51 46 41 36 31 26 -0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

DNA Fragment Size (Log kb)

Figure 2. Calibration curve depicting the migration distance and DNA fragment size in log kilobases. The migration distance was measured using a ruler in milimeters from the well (at the top) of the 0.6% molten agarose (in TRIS acetate EDTA (TAE) buffer) gel electrophoresis chamber, which contained 0.50X TAE buffer in the chamber reservoirs. The DNA fragment size was measured using the band sizes of the DNA ladder as a reference3, ranging from 0.5kb to 10kb. TABLE 2. Calculated size and the true size of enzymatic digestions of Lambda DNA.

True Size of Fragments (kb)4 EcoRI (E) Lambda HindIII (H) Digest 21.225 7.421 5.804 5.643 4.878 3.530

Lambda Digest 23.129 9.416 6.557 4.361 2.322 2.027 0.564 0.125

a

Calculated DNA fragment Size (kb) EcoRI (E) Lambda HindIII (H) Digest 15.018 8.560 7.438 6.463 5.616 4.240

Lambda Digest 16.111 10.947 8.867 6.463 5.616 3.952 3.201

a

The migrated distance of each fragment was measure from the agarose gel image (Figure

1) and the size of each DNA fragment was determined using the equation from the calibration curve (Figure 2). The agarose gel was prepared and loaded as described in the caption of Figure 1.

8. The calculated fragment sizes differ than the published values as seen in Table 2 due to the fact that the calibration curve obtained in Figure 2 was plotted according to the fragment sizes ranging from 0.5kb to 10kb. If the size of the obtained fragments for each Lambda DNA digests were actually larger than 10kb, then we would be extrapolating data from the graph, which would lead to inaccurate results. Since the DNA ladder has a known molecular weight as well as base pairs for the DNA, it may not include the sizes of each of the fragments from each digest of the enzyme on the Lambda DNA, which again, establishes the fact that the calibration curve was not dependable to calculate the DNA fragments.

References 1. Purity Ratios | Denovix https://www.denovix.com/pdf/130-Purity-Ratios.pdf (accessed February 5, 2019).

2. Mayer, M. What Causes Smearing in Electrophoresis? https://sciencing.com/causes-smearing-electrophoresis-6404726.html (accessed February 5, 2019) 3. Nichols, E.R. (2019) Biochemistry II: Catabolism, Synthesis, and Information Pathways Laboratory Manual 4. Restriction Digestion and Analysis of Lambda DNA. Biotechnology Explorer. https://www.cpet.ufl.edu/wp-content/uploads/2013/10/Restriction-Digest-andAnalysis-of-Lambda-DNA-kit-manual.pdf (accessed February 5, 2019)...