Muhammad Rao , 20101006

Final Report Chapter 7

Topic: Plasmid DNA Extraction from Recombinant E.coli Purpose: To provide an understanding of the fundamentals of genetic engineering
through an investigation of the plasmid DNA extraction method for DNA recombination. Theoretical Background
Genetic recombination technology utilizing DNA has been developed since the double helix structure of DNA was clarified by James D. Watson and Francis Crick in 1953. For the last 20 years, prominent advances of recombinant DNA technology have made possible the mass production of medically or industrially useful proteins from virus, yeast, fungi animal, plant or insect cells where once these proteins were only available in extremely small quantities in the natural environment. Although the host cell for useful protein production is selected carefully according to the characteristics and requirements of the protein, the most commonly used protein is E.coli due to the well-organized E.Coli research. The plasmid as vector DNA is used for novel gene insertion into E.coli chromosomal DNA (single copy) has been used in this manner with a size of 4.7 Mbp. The experiment attempts to introduce the method of plasmid extraction from recombinant E coli, which is the most basic technology among genetic engineering technologies. The following two methods are most commonly used.

1) Alkaline denaturation method In this method the characteristics of the different structures between the two types of DNA can be used to separate only the plasmid. Chromosomal DNA is much larger than plasmid DNA and has the linear

form ( non- supercoiled ). Plasmid DNA has a relatively small size and has a covalently closed circular form

Denaturation: If ( NaOH) is added to the two types of DNA to adjust the pH to a range of 12.0-12.5, the chromosomal DNA is denatured from a double strand to a single strand. However, the plasmid DNA is not denatured to a single strand in the PH range of 12.0 12.5. Neutralization: The solution is neutralized to pH 7.0 by adding acid; the chromosomal DNA, denatured to a single strand, is tangled to a very large aggregation which prevents combining single strands into double strands. Centrifugation: The aggregated chromosomal DNA is participated into a pellet by centrifugation. It is then possible to separate the soluble plasmid of DNA from the upper layer. Purification: The protein in the upper layer is eliminated by adding phenol/chloroform. The pure plasmid DNA is then obtained by adding ethanol, which precipitates the plasmid DNA.

2) Ethidium Bromide-cesium chloride density gradient centrifugation If a bottle with Cesium chloride solution is centrifuged at a very high velocity, a density gradient is formed. The DNA, RNA, protein and other materials, which have different position in the density gradient according to the buoyant density. It is possible to separate the DNA using the density gradient. However the different structure of the linear DNA and the supercoiled DNA causes a difference in the decreasing buoyancy, Specifically the linear DNA is located more in the upper part compared to the supercoiled DNA in the CsCl density gradient with the addition of EtBr. Consequently, it can purify the pure supercoiled plasmid DNA from the cell extract.

: 3) Agarose Gel: Agarose gel electrophoresis is a method of gel electrophoresis used in clinical chemistry to separate proteins by charge and or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

Plasmid from purified recombinant DNA can be confirmed by the agarose gel electrophoresis method. The DNA is assigned a (-) charge by phosphate in a backbone; hence it moves from (-) electrode toward (+) electrode, more quickly in case of smaller size.The DNA is agarose can confirm through EtBr staining.As the agarose gel is put into the EtBr solution, EtBr intercalates DNA and sa it destains into water, the EtBr without intercalating DNA is removed an only intercalated DNA remains in the agarose gel.

3) Gene Cloning: This experiment is also very important in Gene cloning because if we can find the plasmid and see the nature of the plasmid in the experiment then we can definitely attach another gene into it and create a new organism of fix the defects in the existing gene. By using gene cloning we can also make a large amount of E.coli and produce a large quantity of protein.

Materials and Tools

1) Recombinant strain: E.coli XLI-Blue (pUC19) 2) Culture tube 3) LB media 10 mL 4) Solution 1 (Resuspension buffer) 50 mM Glucose, 25 mM Tris-Cl(pH8.0), 10 mM EDTA, RNase A solution. 5) Solution 2 (Lysis Butter) 0.2N NaOH , 1% SDS 6) Solution 3 (Neutralization Buffer) 5M potassium acetate 60 m L glacial acetic acid 11.5 mL H2O 28.5 mL 7) Ethanol 8) TE Buffer 9) Agarose 10) TAE Buffer: 0.04 M Tris acetate, I mM EDTA 11) Loading dye (6x): 0.25 % bromophenol blue, 0.25 % xylene cyanol FF, 30% glycerol. 12) Ethidium Bromide 13) DNA molecular size marker 14) Microcentrifuge 15) Micropipette 16) Shaker 17) Vortex 18) Photosystem.

Procedure :
A) Extracting Plasmid DNA 1) Choose a single colony from a freshly streaked bacterial plate and use it to inoculate LB plus an appropriate antibiotic. Incubate the culture overnight while shaking. 2) Harvest 3-5 mL of bacterial culture by centrifugation at 13000 rpm for 30 sec at RT and discard the supernant. 3) Resuspend the pellet in 250uL of resuspension buffer,vortexing until no clumps of the cell pellet remain. 4) Add 250ul of Lysis buffer to the resuspended cells. Close tubes and gently mix by inverting the tube several times. Do not vortex and do not exceed 5 min of lysis time. 5) Add 350ul of neutralization buffer and gently mix by inverting the tube several times. 6) Centrifuge at 13000 rpm for 10 min at 4C.While waiting for the centrifugation, insert a column into the collection tube. 7) After centrifugation, transfer supernant promptly into the column. 8) Centrifuge at 13000 rpm for 60 sec. Remove the column from the collection tube and discard the filtrate in collection tube. 9) Add 700ul of washing buffer B and centrifuge at 13000 rpm for 60 sec. discard the filtrate in the collection tube and place the spin column back into the same collection tube. 10) Centrifuge at 13000 rpm for 60 sec to dry the filter membrane 11) Put the column into a clean and sterile centrifuge tube. Add 50ul of elution buffer or distilled water to the upper reservoir of the column and let it stand for 1 min. B) 1) 2) 3) 4) Gel Electrophoresis: Put 0.32g Agarose in a 50 mL flask with TAE Buffer. Heat moderately and then let it cool and then pour into gel caster. Completely take out the gel and transfer it with the help of gel electrophoresis kit. Prepare the DNA sample with 5uL loading dye(6X) 1uL and then load it on Agarose gel and let it mix. 5) Apply 100 Volts for 30 minutes and after 10 minutes add ethidium Bromide solution. 6) Let the gel de-stain and then take it out and verify the presence of plasmid DNA under UVIlluminator and take a polaroid picture After getting the plasmid DNA from the purification process we do gel electrophoresis to check the purity, concentration, size and molecular weight, that whether we separated the plasmid DNA correctly or there is some contamination in the DNA, if we found any contamination then there is was a problem in step 7 of the procedure.

Data and Results:

Figure: Gel Electrophoresis Agarose Gel Electrophoresis: In the above picture which is obtained from the gel electrophoresis the left two bands are plasmid DNA and as the nature of the DNA is negatively charged, when voltage is applied they move towards the positive charge, On the extreme right hand side is the DNA ladder, Its kind of the two lines trying to keep balance, The bottom is supercoiled state and it has been separated in two. 1) Plasmid Size : 2800bp From the above figure we can most definitely say that the size of the plasmid is 2800bp and we know that 2800 bp plasmids are pUC 19 2686, because the ladder on the top is around 2686 bp (-) pole side and is marked in red in PUC 19 plasmid.

2) Concentration : 4.8 The concentration of the plasmid which is shown in the gel electrophoresis is approximately 4.8 , because as compared with the ladder which is at 43.92 position , compared to the paper the ladder in the diagram looks probably at 3) Analysis Of Lines After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation). The illuminator apparatus mostly also contains imaging apparatus that takes an image of the gel, after illumination with UV radiation. The ethidium bromide fluoresces reddish-orange in the presence of DNA, since it has intercalated with the DNA. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA. The gel can then be photographed usually with a digital or Polaroid camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white.

Discussion:
A nucleic acid material of E.coli is composed of plasmid DNA, chromosomal DNA, and RNA .In this experiment we removed the other components and just obtained the plasmid DNA. First we separated nucleic acids and proteins, then from the protein solution, phenol chloroform layer is removed and the aqueous layer remaining on the floor is also removed. But in the next step when we were clearing chromosomal DNA and to isolate plasmid DNA we used alkaline denaturation. solution2, solution3 is used for this purpose, solution3 (Neutralization Buffer) is used and then centrifuged. The white clotted substance chromosomal DNA and protein become two relieving substances and then chromosomal DNA and protein can be separated. Error Analysis: There might be certain impurities left in the solution this was due to the transferring of a little bit of white pellet during step 7 of the procedure. IF we transferred the white pellet the protein and genome DNA will also transfer to the solution. Plasmid pUC19 investigation of the genetic map :

1) lacZ 'gene - In this gene E.coli decompose in the Lactose encoding the enzyme -galactosidase . 2) ApR gene- Ampicillin resistant gene. (, E.coli are likely to be walked away from the plasmid without this gene) 3) Rep This gene is responsible for the replication of plasmids 4) Lacl Gene - IPTG inducer-free condition gene, lacZ 'gene prevents transcription of the repressor in part of the production.

Multiple Cloning Site (MCS) - The site of various restriction enzymes, the region where the action is recombinant DNA is inserted.

References :
1) Molecular cloning. A Laboratory manual. 2nd ed. (1989) Sambrook J, Fritsch EF, Maniatis T. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2)Gene cloning. 3rd ed. (1995) Brown T.A. Chapman & Hall, London, UK. 3)Principles of gene manipulation. 5th ed. (1994) Old R. W. and Primrose S. B. Oxford, UK. Blackwell Science Ltd,

4) Molecular Biotechnology - principles and applications of recombinant DNA. 3rd ed. R.Glick and Jack J. Pasternak .5)Essentials of Molecular Biology. 4th ed. George M. Malacinski 6)Wikipedia

Bernard