Student Name :NguyễnNhậtThái Student ID : BTIU09026

1. Can we transfer digested plasmid (linear plasmid) into the competent
cell? In the laboratory, we transfer digested plasmid (linear plasmid) into the competent cell by the process of transformation.It can contain some genes that the bacterium would not normally posses. These extra genes can provide a growth advantage to bacteria by providing the gene for an enzyme such as amylase, beta-lactamase. Reference: Genetics A conceptual approach, Third edition, Benjamin A. Pierce Transformation of the Host cells, Virtual & Accessible Laboratories Universalizing Education, sub=3&brch=77&sim=1107&cnt=1

2. Given 3 antibiotics (kanamycine, ampicilline, tetracyline) and Xgal- IPTG.
Design a selection media system to reveal the right colonies.

The first petri dish of LB agar that contains kanamycine and Xgal-IPTG, is used to culture transformed cells. The plasmids (pCambia 2301) contain kanamycine resistance gene, so the bacteria that have success transformed plasmid will alive (low rate) and grow in separated colonies, otherwise they will die. In addition, the plasmid also contains the lac Z which encodes βgalactosidasethat can cleave X-gal and make it become blue color. Then we can reveal the blue one is right colony. The second petri dish of LB agar contains other antibiotics (such as Ampiciline, Tetraciline) is used to culture transformed cells. There is no growing in this dish because the plasmid does not have these antibiotics resistance genes. The third petri dish with LB agar (the negative control) is used for comparison if anything goes wrong.

3. Which are restriction enzymes can be used to digest the plasmid pCambia

this is based on the interaction between negatively charged phosphates of the DNA backbone and positively charged DEAE groups (dimethylaminoethanol)on the surface of the resin.D. Ph. Bernard R.5) 40µM EDTA Wash the containing residual cellular proteins and debris. • Column Wash Solution 55% Ethanol: precipitate DNA 80mM potassium acetate 8. . Department of Molecular and Cellular Biology. Glick and Jack J. along with the cellular debris. denature most of the proteins in the cells • Neutralization Solution 1.There are several restriction enzymes can be used: Enzyme Restriction Site BamHI G•GATCC EcoRI G•AATTC HindIII A•AGCTT PstI CTGCA•G Reference: Molecular Biotechnology Third edition. and column wash solution. Reference: Plasmid DNA Isolation. Pasternak 4.3mM Tris-HCl (pH 7. 100µg/ml RNase A: Degrade cellular RNA • Cell Lysis Solution 0. neutralization solution. • Cell Resuspension Solution 50mM Tris-HCl (pH 7. protects the DNA from degradativeenzymes. 1% SDS: Solubilize the cell membrane.. Nadja Anderson. Elements and functions of cell resuspension solution.8): precipitates the SDS from solution. How does resin work to maintain the plasmid in the membrane? Resin bind to maintain the plasmid.2M NaOH: Denatures the DNA into single strands.The University of Arizona 5.32M potassium acetate (pH 4. cell lysis solution.5): Maintain a constant pH 10mM EDTA: Destabilize the cell membrane.

com/plasmid/anionexchangeresin. Describe how plasmids isolate from cell step by step. What is the white precipitate? The white precipitate is mixture of the single stranded genomic 8. Suzanne. Reference:How Alkaline Lysis Works. Bitesize Bio. . Step 1 : Centrifuge the medium at 10000 rpm for 10 minutes  the pellet is cells that contains plasmids. How does water work to elute plasmid out of the membrane? Water tends to have a low pH (4-5) and high molecular weight DNA may not completely rehydrate in the short time used for elution.aspx 6. Reference: How Silica Spin Column DNA and RNA Preps Work. the SDS and the denatured cellular proteins stick together through hydrophobic interactions.Reference: QIAGEN Anion-Exchange Resin form http://www. June 28. 2010 http://bitesizebio. Elution of DNA can be maximized by allowing the buffer to sit in the membrane for a few minutes before centrifugation. Step 2 : add Resuspension Solution  cells that contains plasmids in the 7. November 07 2007 http://bitesizebio.qiagen.

Why we put glycerol (restriction enzyme) less than 5% of total volume? Because the enzyme storage buffer contains glycerol as antifreeze to allow it to survive at –20°C. Reference: Restriction enzyme digestion of DNA: basic method. Ying Zhai and Qingyu Wang. http://www.Step 3 : add Cell Lysis Solution  Plasmids are dissolved in the medium. Agricultural Division. Explain the conditions of centrifugation (5000 rpm. which could degrade transformation efficiency to a certain extent. 10 min) The factors influencing the efficiency are often related to conditions that render cells competent. some dead bacteria would form sediment together with activity bacteria. Yepeng Sun. Step 5 :add resin. Yan Zhao. Reference:An improved calcium chloride method preparation and transformation of competent cells. Xiaowei Li. Department of Pathology. If Centrifugation time exceeds 10 min. University of Liverpool. 4oC. Yan Zhang. column wash solution and centrifuge  Plasmids are still trapped in the Minicolumn Step 6 : add nuclease-free water thencentrifuge Plasmids are dissolved in water. Matt Lewis. College of Plant Science. Otherwise.methodbook. These incldudeusing cells at a temperature of 4oC harvested during the logarithmic phase of growth thecentrifugation speed and time. Step 4 : add Neutralization Solution then centrifuge  Plasmids are dissolved in the lysate. 9. . Xin Sui. The glycerol will inhibit the digestion if present atmore than 5% of total Jilin University.html 10. Laboratory of Molecular Biology. it was also found that a great deal of active bacteria could form sediment when centrifugation time was five minute.

which forces the DNA to enter the cells either through cell pores or the damaged cell wall. How does heat shock trigger the plasmids enter competent cells? The heat shock creates a thermal imbalance on either side of the cell membrane. In100 mM CaCl2. Why do added CaCl2 twice with different concentration? The transformation efficiency of competent cells increased among the concentration of the CaCl2. Reference: Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. What is function(s) of CaCl2 in competent preparation? The presence of calcium ions makes the cell become permeable to plasmid DNA. The cellular absorption ability of exogenous DNA will be increased after heat shock at 42°C. lipid array on cell membrane is destroyed and then a liquid crystal would be formed. Yan Zhang. S. Yan Zhao. Xin Sui. College of Plant Science. M. Dagert.. Xiaowei Li. 13. Laboratory of Molecular Biology. Agricultural Division. Reference:An improved calcium chloride method preparation and transformation of competent cells. The bacteria would be distended when incubated at 0°C. and DNA in the mixture can be formedinto hydroxyapatite (anti-DNase) then stick to the surface of cells. Ehrlich. (1979) 12. Ying Zhai and Qingyu Wang. .11. Yepeng Sun. The plasmid DNA can then pass into the cell upon heat shock. Jilin University. hypotonic calcium chloride solution. positively charged Ca2+ attract both the negatively charged DNA backbone and the negatively charged groups in the Lipopolysaccharide inner core.

in/? sub=3&brch=77&sim=1107&cnt=2 .vlab. http://amrita.Reference: Transformation of the Host cells. Virtual & Accessible Laboratories Universalizing

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