Experiment 9 Extraction of plasmid from bacteria

1. Experiment 9 Extraction of plasmid from bacteria

2. Plasmid Definition：A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA.1 They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre ring in Saccharomyces cerevisiae).

3. Plasmid Application：Serve as gene vector to carry the target gene into host cells, is an important tool for gene cloning

4. Application of plasmid • To serve as vector to transfer target gene into bacteria and to amplify the target gene in host cells, and to produce a lot of copies of target gene • To transfer target gene into host cells, not only amplify target gene, but also to make target gene express and to produce the protein coded by the target gene

5. Application of plasmid • Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. • Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria that take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way, the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested, and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.

6. Application of plasmid • Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics. • However, a plasmid can contain inserts of only about 1–10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are used.

7. Conformation of plasmid Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:

8. Conformation of plasmid From slowest to fastest: • "Nicked Open-Circular" DNA has one strand cut. • "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). • "Linear" DNA has free ends, either because both strands have been cut or because the DNA was linear in vivo. • "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. • "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

10. Basic Strategy of Gene Cloning To get target gene Select proper plasmid Recombination in vitro Recombinant DNA Transformation Competetive cells Screening To identify the positive clones Amplification A lot of copies of the recombinant DNA

11. The method to extract plasmid from bacteria Alkaline Lysis Method

12. Major Steps for the alkaline lysis method 1.To culture the bacteria containing plasmids 2. To harvest and lysis the bacteria 3. To extract and purify the plasmid DNA 4. To identify the plasmid DNA

13. Principle Under alkaline solution, chromosome DNA and plasmid DNA also be denatured, but plasmid DNA still remain twist. When the solution pH be regulated to neutral state meanwhile with high salt and lower temperature, chromosome DNA, macromolecular RNA and proteins would be precipitated by SDS, plasmid DNA keep soluble in water. By centrifuging, plasmid DNA could be seperated from a majority of cell fragments, chromosome DNA, proteins. Finally, plasmid DNA could be purified with chloroform, phenol so on.

14. Protocol 1. To add 1.5 ml of bacteria solution after over one night culture into EP, centrifuge one minute by 10 000r/min, discard the supernatant solution. 2. To add 100 μl ofsolutionⅠ，strongly shake the EP tube to let the thalli be suspend evenly again, stay it for 5 minutes in room temperature. 3. To add 200 μl ofsolutionⅡ（immediately used as soon as preparation），gently overturn several times to mix well, stay it for 5 minutes in room temperature. 4. To add 150μlof precooledsolution Ⅲ ，gently overturn several times (4~6 times), stay on ice for 10 mintues.

15. Solution I：50 mmol/L glucose, 25 mmol/L Tris-Cl，pH 8.0 10 mmol/L EDTA， pH 8.0 Solution II： 0.2 mol/L NaOH, 1% SDS Solution III：29.4gof potassium acetate， 11.5 ml of ice acetate， H2O to be added to 100ml。

16. Protocol 5. 12 000 r/mcentrifuge 15 min( if the supernatant is still turbid, it is possible to transfer the supernatant to a new EP tube, and centrifuge 5 min again). To transfer the supernatant into a new EP tube, add the same volume of phenol: chloroform: isoamyl alcohol (25:24:1）extraction one time（12 000r/min centrifuged 10min). 6. Absorb the supernatant carefully to another new EP, to add 2.5 times of enthanol, mix well, stay in room temperture 10 min, centrifuge 10 min for 12 000r/min, to precipitate the plasmid DNA.

17. Protocol 7. Wash the precipitate two times with 70% alcohol, spontaneously dryness 8. To solubilize the precipitate DNA into 30~50 μl ddH2O，conserve under -20℃ for standby.

18. pGEM-T Easy Vector Plot

21. Demand：2persons as one group To consider the action of every reagent during the experiment process