Lab Report #2 Purification of Plasmid DNA from E.coli cells PDF

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Complete Lab Report for Biochemistry on purification of plasmid DNA...

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Purification of plasmid DNA from E.coli cells Michael J. Blanco September 26, 2016

Abstract Plasmid pBR322 was purified from an E.coli cell using a Qiagen kit. The Qiagen kit contains the necessary buffers and instruction to purify plasmid pBR322. The plasmid pBR322 contain 4,361 base pair. Through agarose gel electrophoresis we compare the purify plasmid with the prepared marker and calculated the base pair with 1.99% error. Introduction Plasmid purification is used to isolate and purify DNA from genomic DNA, proteins, ribosomes, and bacterial cell well, such as, E.coli . Plasmids are circular double stranded DNA molecules that occur naturally in bacteria and come in variety of sizes. Plasmids carry genes for the inactivation of antibiotics, production of toxins, and breakdown of natural product. A common plasmid-cloning vector in E.coli is pBR322. The molecule is double stranded circle

Figure 1-pBR322 Vector

with 4,361 base pairs length (Watson ,1988). It also contains the genes for resistance to ampicillin and tetracycline as it shows in Figure 1 (pBR322 Vector). The molecular weight of the plasmid is approximately 2.83 x106 Daltons (Biolabs).

The purification of a plasmid is based on the alkaline lysis of E.coli cells. There are three step involve in the purification of DNA plasmid. The steps involved is preparation of bacterial lysate, adsorption of DNA, and washing and elution of plasmid DNA (Zhang, Cahalan). The plasmid purification is important for molecular cloning, as well as, DNA isolation. The use of buffers is important to the purification of the plasmid. A total of 5 buffers are use for the purification of the plasmid: P1, P2, N3, PE, and EB. P1 buffer re-suspend the cells and hydrolyze RNA. The P2 buffer breaks open or lyse the cell wall, however if it is left too long it will cleave to the DNA. The N3 buffer denature the protein and the PE buffer keeps the plasmid on the column and washes it. The final buffer is EB, which elute the DNA from the silica gel. A suitable technique to separate macromolecules based on size is electrophoresis. Electrophoresis is carried out in porous gel, such as, agarose to enhance separation (Berg, Tymoczko, Gatto, & Stryer, 2015). Electric field is applied to the electrophoresis chamber where the protein can migrate from negative to positive electrode (Berg, Tymoczko, Gatto, & Stryer, 2015). The separation is based on the size of the molecule relative to the pore of the gel (Berg, Tymoczko, Gatto, & Stryer , 2015). Procedure E.coli cell was first obtained in three separate 2 mL micro centrifuge tubes. Each tube had 1.5 mL of the E.coli cell; in total there was 4.5 mL of E.coli cell. The micro centrifuge tube was centrifuged for 10 minutes at maximum

speed. The supernatant was discarded from all three tubes and the pellets were combined and re-suspended with 250 μL of P1 and P2 buffers. The mixture was mixed thoroughly. Then 350 μL of N3 buffer was added, mixed, and centrifuge for 10 minutes at maximum speed. After the centrifugation, 800 μL of the supernatant was added to a spin column and centrifuged at same time and speed as prescribed above. The spin column was washed with 750 μL of PE buffer and was centrifuged again. The spin column was placed in a new clean 2 mL centrifuge tube. An addition of 50 μL EB buffer was added to the spin column and held stationary for 1 minute and centrifuged for another minute. The buffer content is: P1: 50 mM Tris/HCl, pH 8, 10mM EDTA, 0.1 mg/mL RNase P2: 200 mM NaOH, 1% SDS N3: .9 M at pH 4.8 acetate buffer PE: 10 mM Tris/HCl pH 7.8, 80% ethanol EB: 10 mM Tris/HCl pH 8.5 A 1% agarose gel solution for electrophoresis was prepared. The solution contained .5g of agarose in 50 mL of 1mM Tris/Borate/EDTA (TBE). The TBE was diluted to obtain the required concentration. The solution was microwaved for 2 minutes into electrophoresis chamber and hardened for approximately 30 minutes. The plasmid sample was prepared with 3.3 μL 6x dye, 6.7 μL EB buffer, and 10 μL of the plasmid. The sample was loaded into the well of the gel.

Results

Log(DNA BasePairs)

Log(DNA size) vs. Migration Distance 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

f(x) = − 0.18 x + 5.17 R² = 0.93

5

6

7

8

Migration Distance (cm)

9

10

11

The equation above can be used to determine the number of base pair that our purified plasmid contains. y = -.1813x + 5.1711 The distance of the plasmid band from the well is 8.4 cm. Plug into x: y= -.1813 (8.4) + 5.1711 y= 3.64818 Since the above answer is the log (DNA Base pairs). DNA base pairs was solved. 103.64818 = 4448 base pairs The pBR322 plasmid contains 4,361 base pairs. Percent Error: ⌈ 4361−4448 ⌉ x 100 % 4361

= 1.99%

Discussion Plasmid Purification is an effective technique to isolate and purify DNA and important for molecular cloning. The pBR322 plasmid was purified form E.coli cell using Qiagen kit. The Qiagen kit contain all the necessary buffer to purify the plasmid, such as, P1, P2, N3, PE, and EB buffers. The Qiagen kit is simple and requires minimal effort to perform the purification, however, if the purification did not go as expected it would be difficult to determine the problem. The purified plasmid’s base pairs were determined through electrophoresis by comparing to the marker. Electrophoresis helps separate macromolecules like DNA. DNA fragments normally separate according to their size. The electrophoresis contains agarose gel that is a thick and viscous substance which attracts negatively charges DNA molecules. The DNA molecule

are pulled to the positive end of the gel base by current, however encounter resistance from the agarose gel. Our results indicate that plasmid pBR322 contains 4,448 base pairs compared to the literature value of 4,361 base pairs (Watson, 1988). There is a 1.99% error. The error could have resulted from the difficulty to measure the migration distance, accurately. There are some weak bands on the plasmid lane, such as, lane 1 and lane 2. This could be as a result of losing some DNA during digestion and gel extraction.

References

1. Berg, J. M., Tymoczko, J. L., Gatto, G. J., & Stryer, L. (2015). Biochemistry. 71-73. 2. Biolabs, N. E. pBR322 Vector https://www.neb.com/products/n3033-pbr322vector (accessed Sep 25, 2016). 3. pBR322 Vector https://www.promega.com/products/biochemicals-andlabware/nucleic-acids/pbr322-vector/ (accessed Sep 25, 2016). 4. Watson, N. Gene 1988, 70 (2), 399–403. 5. Zhang, S.; Cahalan, M. Purifying Plasmid DNA Using the Qiagen Miniprep Kit | Protocol http://www.jove.com/video/247/purifying-plasmid-dna-frombacterial-colonies-using-qiagen-miniprep (accessed Sep 25, 2016)....