SECTION OF BIO-ENGINEERING TECHNOLOGY, UniKL MICET.

LAB 1: PLASMID EXTRACTION (Using Alkaline Lysis Minipreps Method)

Introduction Many different methods are available for isolation of plasmid DNA. Nowadays, commercial kit in which utilizes an affinity column is commonly used to purify the plasmid DNA as they are less laborious and less time consuming. In this practical however, students are required to extract plasmid using conventional method whereby plasmid DNA is precipitated using ethanol instead of affinity column. To isolate plasmid DNA from bacteria (E. coli in this case) containing plasmid, the bacteria is grown from a single colony in a suitable medium such as Luria Broth (LB). An aliquot of the culture is used for preparing the plasmid DNA.

Methodology 1. Transfer 2 ml overnight culture into 2 ml microtube. 2. Spin down the cells at 13 000 rpm in microcentrifuge for 1 minutes. Discard supernatant. 3. Resuspend pellet in 200 ul GET (50 mM glucose, 10 mM EDTA, 25 mM Tris HCl, pH 8.0) solution, vortex vigorously and incubate on ice for 5 minutes. 4. Add 100 ul of 2% SDS followed by 100 ul of 0.4M NaOH. Mix gently. Place on ice for 5 minutes. 5. Add 150 ul of 3 M sodium acetate, pH 5.2. Vortex for 10 s. Place on ice for 5 minutes. 6. Centrifuge at 13 000 rpm in microcentrifuge for 5 minute. Transfer the supernatant to a clean microtube. 7. Add 2 volumes of absolute ethanol to the supernatant, mix and incubate at room temperature for 2 minutes. 8. Pellet the nucleic acid by centrifuging at 13 000 rpm, for 10 min at 4oC. Discard the supernatant. 9. Wash the pellet with 500 ul of 70% cold ethanol and keep on ice for 2 min. Spin at 13000 rpm for 5 min and discard the supernatant. 10. Air dries the pellet for 15 min before resuspend in 30 ul of sterile distilled water. 11. Add 4 ul of a 20 ug/ml solution of RNAase and incubate for 20 minutes at 37oC.

12. Run the plasmid DNA on agarose gel electrophoresis with appropriate DNA marker. Questions 1. State the function of each compound in the following solution: Solution GET : Glucose EDTA SDS NaOH Na acetate Absolute ethanol 70% ethanol RNAase Function

a.

b.

c. d. e. f.