Practical Of Genetics

Transcription

1 Practical Of Genetics

2 To learn how to extract plasmid DNA from E.coli and to observe the analysis of plasmid DNA by gel electrophoresis.

3 Over the past decades it became evident that virtually in all bacterial species plasmids exist. These accessory genetic elements are defined as autonomously replicating extrachromosomal DNA.

4 A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded.

5 A chromosome is an organized structure of DNA and protein that is found in cells.

6 Plasmids E. coli Chromosome Electron micrograph of DNA from a lysed E. coli cell

7 Plasmids typically account for only a small fraction of a bacterial genome corresponding roughly to a range between 1 and 200 kilobase pairs. However, extremely large megaplasmids with sizes far beyond 200kb have also been detected such as in Rhizobium and others. Plasmids of more than 50 kb might be characterized as large plasmids whereas plasmids used as tools in molecular genetics are often smaller than 10 kb.

8 Plasmid DNA can be separated from the rest of the bacteria DNA because it is much smaller circles than the circular bacterial chromosome. The separation of the two forms of DNA is based on Most procedures involve in which the bacterial membrane is perforated and the contents released.

9 In this exercise, we will be using method for the isolation of plasmid DNA from different E. coli strains.

10 In the process, the long circular chromosome become sheared into linear fragments but the smaller plasmids remain intact. Intact plasmid can be separated from linear DNA because they remain supercoiled.

11 The lysis buffer contains sodium hydroxide and SDS, the purpose of which is to completely denature of the plasmid and genomic DNA (i.e. separate the DNA into single strands). It is critical that this step is performed quickly because too long in the denaturing conditions of this solution may result in irreversibly denatured plasmid at the end.

12 Next the sample is neutralized in a potassium acetate solution to renature the plasmid. And this is the key to the separation of the plasmid and genomic DNA. Because plasmid is small, it can easily reanneal. But the genomic DNA is too long to reanneal properly and instead it becomes tangled so the complimentary strands stay separated.

13 When the sample is centrifuged, the genomic DNA is still bound to protein and gets pulled down while plasmid DNA is soluble and free. It is key at this step not to vortex or mix the sample vigorously because the genomic DNA is easy to break, and broken genomic DNA can be small enough to re-anneal and go into solution with the plasmid. The plasmid DNA is recovered in the supernatant and can now be ethanol precipitated.

14 In this laboratory exercise, three types of solutions are used: Solution I: Glucose EDTA tris (ph8.0) RNase Solution II NaOH (ph12) SDS potassium acetate Solution III glacial acetic acid (ph 5.4) H2O

15 In this laboratory exercise, the E. coli bacterial cells are resuspended in buffer containing : gives osmotic shock that leads to the rupture of cell wall and membrane glucose EDTA RNase stabilizes the cell membrane by binding divalent cations (Mg++,Ca++) and inhibits nuclease activity destroys the cell s RNA The cell membrane lyses, releasing the cell contents

16 SDS dissolves the phospholipid and protein components of the cell membrane NaOH denatures both plasmid and chromosomal DNA into single strands Chromosomal DNA separates completely into individual strands The single-stranded plasmid loops remain linked together like interlocked rings.

17 potassium acetate forms an SDS/lipid/protein precipitates glacial acetic acid neutralizes the alkaline ph (NaOH) At neutral ph, genomic DNA renatures and is trapped in the SDS/lipid/protein precipitate. The plasmid DNA renatures into double-stranded molecules that remain in solution

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19 1. grow up a 3 ml overnight of E. coli broth media Luria Broth (LB). 2. pour 1.5 ml into an eppendrof tube and Pellet cells by centrifuging at 12,000 rpm for 2 minutes. 3. Pour off supernatant and Add another 1.5 ml of culture to the same tube and centrifuge 2 min and pour off the supernatent. 4. Resuspend the pellet by briefly vortex in 250µl of solution I(stored at 4ºC ).

20 5. Add 250 µl of solution II (stored at RT). Mix GENTLY by inverting and rotating the tube several times. DO NOT vortex. Put on ice 5 minutes. 6. Add 350 µl of cold solution III (stored at 4 º C). 7. Mix by inverting the tube 6-8 times. Put on ice for 5 minutes. 8. Spin 12 minutes at rpm in a microcentrifuge at 4º C. (Pellet contains: PDS, Lipids, Proteins and Chromosomal DNA).

21 Separate plasmid DNA from contaminants by centrifugation Supernatant contains: - Plasmid DNA - Soluble cellular constituents Pellet contains: - PDS - Lipids - Proteins - Chromosomal DNA

22 9. Transfer 350 µl of supernatant (contains plasmid DNA) to a fresh tube. 10. Add 2 volumes ethanol at room tempreture. Mix and stand for 2min at RT. 11. Pellet 5 min in microcentrifuge at RT. Dry, then resuspend in 50 µl TE. 12. Analyze your preparations using agarose gel elctrophoresis by mix 8µl of the plasmid sample with 2µl of loading buffer or store at 20º C.

23 Harvest cells by centrifugation Spin ~5,000 rcf E. coli culture (cloudy) Supernatant (clear) Pelleted cells Discard supernatant Residual media may interfere with downstream steps Resuspend cells in buffer Thoroughly resuspend cells, making sure that no clumps remain. P1 buffer contains: Tris-Cl (buffering agent) EDTA (metal chelator) RNase A (degrades RNA)

24 Lyse cells with SDS/NaOH solution Adding buffer P2 causes solution to become viscous 1. Sodium dodecyl sulfate Dissolves membranes Binds to and denatures proteins 2. NaOH Denatures DNA Because plasmids are supercoiled, both DNA strands remain entangled after denaturation

25 Neutralize NaOH with potassium acetate solution Mixing with buffer N3 causes a fluffy white precipitate to form. 1. Potassium acetate / acetic acid solution Neutralizes NaOH (renatures plasmid DNA) Converts soluble SDS to insoluble PDS sodium dodecyl sulfate (SDS) potassium dodecyl sulfate (PDS) (H 2 O sol. = 10%) (H 2 O sol. < 0.02%) Wash pellet with 70% EtOH (to remove salts) Dissolve pellet with TE (or other aqueous solution)

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27 PureYield Plasmid Miniprep System Includes: Cell Lysis Buffer (CLC) (Blue) Neutralization Solution (NSC) Endotoxin Removal Wash (ERB) Column Wash Solution (CWC) Elution Buffer (EBB) PureYield Minicolumns PureYield Collection Tubes

28 1. grow up a 3 ml overnight of E. coli broth media Luria Broth (LB). 2. pour 1.5 ml into an eppendrof tube and Pellet cells by centrifuging at 12,000 rpm for 30 seconds. 3. Pour off supernatant and Add another 1.5 ml of culture to the same tube and centrifuge 30 seconds and pour off the supernatent.

29 4. Add 100μl of Cell Lysis Buffer, and mix by inverting the tube 6 times. The solution should change from opaque to clear blue, indicating complete lysis. Note: Proceed to Step 5 within 2 minutes. 5. Add 350μl of cold (4 8 C) Neutralization Solution, and mix thoroughly by inverting the tube. The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization.

30 6. Centrifuge at maximum speed in a microcentrifuge for 3 minutes. 7. Transfer the supernatant (~900μl) to a PureYield Minicolumn. 8. Place the minicolumn into a PureYield Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds. 9. Discard the flowthrough, and place the minicolumn into the same PureYield Collection Tube. 10. Add 200μl of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds. It is not necessary to empty the PureYield Collection Tube.

31 11. Add 400μl of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds. 12. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30μl of Elution Buffer directly to the minicolumn matrix. Let stand for 1 minute at room temperature. 13. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. 14. Analyze your preparations using agarose gel elctrophoresis by mix 8µl of the plasmid sample with 2µl of loading buffer or store at 20º C.

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