Aim: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation

Principle:

Plasmids are double stranded, circular, self-replicating extra chromosomal DNA molecules. They are commonly used as cloning vectors in molecular biology. Alkaline lysis is one of the most commonly used methods for lysing bacterial cells prior to plasmid purification Alkaline lysis method for rapid purification of plasmids exploits the topological difference between plasmid circles and linear chromosomal fragments. Cells are lysed by treating with alkali (NaOH) and a detergent, sodium dodecyl sulphate (SDS). SDS denatures bacterial proteins and NaOH denatures the plasmid and chromosomal DNA. However, in the case of plasmids the strands remain closely circularized since they are linked by the interwined backbones of double helix. In contrast, strands of linear/nicked DNA are free to separate completely. When this mixture of denatured plasmid and chromosomal DNA is neutralized by the addition of sodium or potassium acetate, renaturation occurs. The high salt concentration causes potassium dodecyl sulfate (KDS) to precipitate, and denatured proteins, chromosomal DNA, and cellular debris are coprecipitated in insoluble salt-detergent complexes that can be removed from lysate by centrifugation. Plasmid DNA, being circular and covalently closed, renatures correctly and remains in solution can be precipitated using ethanol/isopropanol. Plasmid DNA may be isolated from small scale (Mini preparation: 1-2 ml) bacterial cultures by treatment with alkali and SDS. The resulting DNA preparation may be screened by electrophoresis or restriction endonuclease digestion.

REQUIREMENTS: Alkaline Lysis Solution I [GTE solution] 50 mM glucose 25 mM Tris-CI (pH 8.0) 10 mM EDTA (pH 8.0) Prepare Solution I from standard stocks in batches of ~ 100 ml, autoclave for 15 minutes at 15 psi (1.05 kg/cm2 ) on liquid cycle, and store at 4oC.
(Note: A buffer which maintains pH, preventing immediate lysis of cells and ensure that bacteria are resuspended completely leaving no cell clumps in order to maximize the number of cells exposed to the lysis reagents.)

Alkaline Lysis Solution II (NaOH/SDS soln) 0.2 N NaOH (freshly diluted from a 10 N stock) 1 % (w/v) SDS Prepare Solution II fresh and use at room temperature.

(Note: An alkali & a detergent that disrupts cell membrane & denatures chromosomal & plasmid DNA.)

Alkaline Lysis Solution III (pH 4.8) 5 M potassium acetate 60.0 ml Glacial acetic acid 11.5 ml H20 28.5 ml The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 40C and transfer it to an ice bucket just before use.
(Note: A buffer which renatures the plasmid DNA.)

70% and 100% ethanol TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0 Gel Loading Dye (6x)
3ml glycerol (30%) 25mg bromophenol blue (0.25%) dH2O to 10mL

50x TAE buffer 242g of Tris base 57.1 ml of Glacial acetic acid 10ml of 0.5M EDTA (pH 8.0)

For 100mL 50x TAE buffer: 24.2 g TRIS (base) 5.71 mL glacial acetic acid 10.0 mL 0.5 M EDTA (14.6 g/100 ml)

Agarose

Procedure: 1. Pour l.5 ml of the bacterial culture containg the plasmid culture into a microfuge tube. Centrifuge at

12,000 rpm for 2mins at 4c. 2. When centrifugation is complete, remove the medium by aspiration, leaving the bacterial pellet as dry as possible. 3. Resuspend the bacterial pellet in 100l of ice-cold Alkaline lysis solution I by vigorous vortexing. 4. Add 200l of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube

on ice for 5 -10mins.

5. Add 150l of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III
through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 15-20 minutes. 6. Centrifuge the bacterial lysate at 10,000 rpm for 5 minutes at 4C in a microfuge. Transfer the supernatant to a fresh tube. 7. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 5 minutes at room temperature. 8. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4C in a microfuge. 9. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4C in a microfuge. The pellet was air dried till all the ethanol has evaporated completely. 10. Dissolve the nucleic acids in 20-50l of TE (pH 8.0). 11. Meanwhile, 1% agarose gel was prepared in 1x TAE buffer. 12. 2 l of gel loading buffer (6x) was added to 12 l of plasmid DNA isolated and was loaded on the gel and electrophoresis was run at 100 volts for approx. 45 minutes. 13. The gel was analysed under UV transilluminator.

Growth of Bacteria and Amplification of Plasmids Plasmid DNA can be isolated from bacterial culture, which is grown in a liquid medium containing appropriate antibiotic. The bacterial culture should be grown in LB medium, inoculated with a single colony picked from an agar plate. For low-copy plasmids like pBR322, chloramphenicol is to be added after the culture attains late-log phase (A600=0.6) and shaken vigorously for several hours to amplify the plasmid. However, for very high-copy-number plasmids like pUC-series plasmids, such amplification is not required.

1
Open circular Linear/nicked Supercoiled

4
23,130 9416 6557 4361 2322 2027

Lane 1: Plasmid pUC19 DNA (Control) 2686bp Lane 2: Plasmid from E.coli transform by pUC18 ed Lane 3: Plasmid from E.coli transform by pUC19 ed Lane 4: Lambda DNA Hind III digest