Student Name :NguynNhtThi Student ID : BTIU09026

MOLECULAR GENETICS LAB REPORT

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, http://amrita.vlab.co.in/? 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 galactosidasethat 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
2301?

There are several restriction enzymes can be used: Enzyme Restriction Site BamHI GGATCC EcoRI GAATTC HindIII AAGCTT PstI CTGCAG Reference: Molecular Biotechnology Third edition, Bernard R. Glick and Jack J. Pasternak 4. Elements and functions of cell resuspension solution, cell lysis solution, neutralization solution, and column wash solution. Cell Resuspension Solution 50mM Tris-HCl (pH 7.5): Maintain a constant pH 10mM EDTA: Destabilize the cell membrane, protects the DNA from degradativeenzymes. 100g/ml RNase A: Degrade cellular RNA Cell Lysis Solution 0.2M NaOH: Denatures the DNA into single strands. 1% SDS: Solubilize the cell membrane; denature most of the proteins in the cells Neutralization Solution 1.32M potassium acetate (pH 4.8): precipitates the SDS from solution, along with the cellular debris. Column Wash Solution 55% Ethanol: precipitate DNA 80mM potassium acetate 8.3mM Tris-HCl (pH 7.5) 40M EDTA Wash the containing residual cellular proteins and debris.

Reference: Plasmid DNA Isolation, Nadja Anderson, Ph.D., Department of Molecular and Cellular Biology,The University of Arizona

5. How does resin work to maintain the plasmid in the membrane?

Resin bind to maintain the plasmid, 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.

Reference: QIAGEN Anion-Exchange Resin form http://www.qiagen.com/plasmid/anionexchangeresin.aspx

6. What is the white precipitate?

The white precipitate is mixture of the single stranded genomic DNA, the SDS and the denatured cellular proteins stick together through hydrophobic interactions.

Reference:How Alkaline Lysis Works, Bitesize Bio, November 07 2007 http://bitesizebio.com/articles/the-basics-how-alkaline-lysis-works

7. 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. Elution of DNA can be maximized by allowing the buffer to sit in the membrane for a few minutes before centrifugation.

Reference: How Silica Spin Column DNA and RNA Preps Work, Suzanne, June 28, 2010 http://bitesizebio.com/articles/how-silica-spin-column-dna-and-rna-preps-work

8. Describe how plasmids isolate from cell step by step.

Step 1 : Centrifuge the medium at 10000 rpm for 10 minutes the pellet is cells that contains plasmids. Step 2 : add Resuspension Solution cells that contains plasmids in the medium.

Step 3 : add Cell Lysis Solution Plasmids are dissolved in the medium. Step 4 : add Neutralization Solution then centrifuge Plasmids are dissolved in the lysate. Step 5 :add resin, column wash solution and centrifuge Plasmids are still trapped in the Minicolumn Step 6 : add nuclease-free water thencentrifuge Plasmids are dissolved in water.

9. 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 20C. The glycerol will inhibit the digestion if present atmore than 5% of total volume.

Reference: Restriction enzyme digestion of DNA: basic method, Matt Lewis, Department of Pathology, University of Liverpool, http://www.methodbook.net/dna/restrdig.html

10. Explain the conditions of centrifugation (5000 rpm, 4oC, 10 min)

The factors influencing the efficiency are often related to conditions that render cells competent. These incldudeusing cells at a temperature of 4oC harvested during the logarithmic phase of growth thecentrifugation speed and time. If Centrifugation time exceeds 10 min, some dead bacteria would form sediment together with activity bacteria, which could degrade transformation efficiency to a certain extent. Otherwise, it was also found that a great deal of active bacteria could form sediment when centrifugation time was five minute. Reference:An improved calcium chloride method preparation and transformation of competent cells, Xiaowei Li, Xin Sui, Yan Zhang, Yepeng Sun, Yan Zhao, Ying Zhai and Qingyu Wang, Laboratory of Molecular Biology, College of Plant Science, Agricultural Division, Jilin University.

11. What is function(s) of CaCl2 in competent preparation?

The presence of calcium ions makes the cell become permeable to plasmid DNA, positively charged Ca2+ attract both the negatively charged DNA backbone and the negatively charged groups in the Lipopolysaccharide inner core. The plasmid DNA can then pass into the cell upon heat shock.

Reference: Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells, Dagert, M.; Ehrlich, S. (1979)

12. Why do added CaCl2 twice with different concentration?

The transformation efficiency of competent cells increased among the concentration of the CaCl2. In100 mM CaCl2, lipid array on cell membrane is destroyed and then a liquid crystal would be formed. The bacteria would be distended when incubated at 0C, hypotonic calcium chloride solution, and DNA in the mixture can be formedinto hydroxyapatite (anti-DNase) then stick to the surface of cells. The cellular absorption ability of exogenous DNA will be increased after heat shock at 42C.

Reference:An improved calcium chloride method preparation and transformation of competent cells, Xiaowei Li, Xin Sui, Yan Zhang, Yepeng Sun, Yan Zhao, Ying Zhai and Qingyu Wang, Laboratory of Molecular Biology, College of Plant Science, Agricultural Division, Jilin University.

13. How does heat shock trigger the plasmids enter competent cells?
The heat shock creates a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells either through cell pores or the damaged cell wall.

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