DNA Plasmid Isolation By Alkaline Lysis Method

OBJECTIVES

Purify recombinant DNA plasmids from the overnight culture.
Small scale plasmid preparations

Separation of Plasma and Serum from Whole Blood

Buffers and Solutions

Alkaline lysis solution I: 50 mM glucose,
25 mM Tris-Cl (pH 8.0), 10 mM
EDTA (pH 8.0),
Deion water Alkaline lysis solution II:
0.2 N NaOH, 1% (w/v) SDS,
de-ion water Alkaline lysis solution III:
5 M potassium acetate,
glacial acetic acid,
de-ion water Ethanol 70% (v/v) Isopropanol TE-RNAase pH 8.0

Method

1. Pour overnight grown culture to 1.5 mL labeled falcon tube.
2. Centrifugate at 14.000 rpm for 1 min.
3. Remove the supernatant from the tube.
4. Repeat step 1-3, until leaves bacterial pellet as dry as possible.
5. Add 150 µL resuspension buffer, resuspend the bacterial pellet properly by vortexing.
6. Add 200 µL lysis solution to bacterial suspension (freshly made), close the tube tightly and mix contents thoroughly by inverting the tube 4-6 times until the solution becomes viscous.
7. Add 300 µL neutralization solution and mix contents thoroughly by inverting the tube 4-6 times.
8. Centrifuge at 14.000 rpm for 5 min.
9. Take the supernatant and transfer to a new 1.5 mL falcon max 300 µL.
10. Add equal volume of isopropanol in the supernatant (300 µL) and mix it by inverting the tube couple of times.
11. Incubate in -80°C for 30 min.
12. Centrifuge at 14.000 for 5 min.
13. Remove the supernatant and add 600 µL EtOH 70%.
14. Centrifuge at 14.000 for 5 min.
15. Remove the supernatant and dry the pellet for 10-30 min.
16. Dissolve the pellet in 20-50 µL TE-RNAase pH 8.0. Confirm the plasmid with 5 µL DNA solvent by Agarose Electrophoresis.

Prepare Of Alkaline Lysis Solution

Alkaline lysis solution I 1. 1 M glucose stock solution (50 mL) a.
Dissolve 9 gram of glucose in 50 mL sterilized de-ion water.
b. Filter sterilize using membrane millipore (0.20 µM).
c. The glucose solution is ready to use or store at 4°C cabinet for preservation.
2. 1 M Tris-Cl stock solution (50 mL) a.
Dissolve 6.057 gram of Tris base in 50 mL sterilized de-ion water.
b. Adjust the pH to the desired value by adding concentrated HCl. 3. 0.5 M
EDTA stock solution (100 mL) a.
Dissolve 14.612 gram of EDTA in 100 mL sterilized de-ion water. b.
Adjust the pH to 8.0 with NaOH

CHARACTERIZATION OF CELL LINES

Alkaline lysis solution I

Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes at 15 psi and store at 4°C.

Alkaline lysis solution I	Volume
1 M Glucose	5 mL
1 M Tris-Cl	2.5 mL
0.5 M EDTA	1 mL
De-ion water	90.5 mL
Total volume	100 mL

Alkaline lysis solution II

1. 10 N NaOH stock solution (50 mL) Dissolve 20 gram of NaOH in 50 mL sterilized de-ion water.
2. 1% (w/v) SDS stock solution (30 mL) Dissolve 0.3 gram of SDS in 30 mL sterilized de-ion water.
Prepare Solution II fresh and use at room temperature.

Alkaline lysis solution II	Volume
0.2 N NaOH	200 µL
1% SDS	1 mL
De-ion water	8.8 mL
Total volume	100 mL

Alkaline lysis solution III

1. 5 M potassium acetate stock solution (100 mL)
Dissolve 49.071 gram of potassium acetate in 100 mL sterilized de-ion water.
Store the solution at 4°C and transfer it to an ice bucket just before use

Alkaline lysis solution III	Volume
5 M Potassium acetate	60 mL
Glacial acetic acid	11.5 mL
De-ion water	28.5 mL
Total volume	100 mL

Translation In Prokaryotes And Eukaryotes

Result…

This is dependent on how the purified plasmids are visualized and characterized, however, a small white pellet should be seen after the ethanol precipitation and centrifugation. A purified plasmid should give a single band on an agarose gel, however, larger bands may be visualized due to super-coiling or nicking. A small bright band will also be seen which is RNA.

BACKGROUND

A very common technique in molecular biology is commonly referred to as “minipreps”, which usually use an alkaline lysis method. Minipreps are used to isolate small quantities of DNA from bacterial colonies to screen colonies for the correct DNA plasmid.
Specific protocols for alkaline lysis differ from laboratory to laboratory, however, they are all based on the same principal. The first stage is to grow the selected bacterial colonies in a small volume (3-5ml) of LB broth containing the selection antibiotic. The bacteria are pelleted and resuspended in a resuspension buffer. This buffer is often a basic pH Tris buffer, which helps to denature DNA and EDTA (ethylenediaminetetraacetic acid) that binds divalent cations destabilizing the membrane and inhibiting DNases (enzymes that degrade DNA). In addition, RNases are also added to degrade the released RNA.

PHYSICAL CELL SEPARATION

Next, the bacteria are lysed with strong alkali (Sodium Hydroxide (NaOH)) and detergent (Sodium Dodecyl Sulfate (SDS)). The SDS detergent solubilizes the phospholipids and proteins of the cell membrane resulting in cell lysis and the release of the contents of the cell. The high concentration of sodium hydroxide denatures the genomic and plasmid DNA, as well as cellular proteins. The cellular DNA becomes linearized and the strands are separated, whereas the plasmid DNA is circular and remains topologically constrained (the two strands, although denatured remain together).
Finally, a neutralization buffer of potassium acetate is added to neutralize the strongly alkaline conditions. The addition of potassium acetate results in a high salt concentration that leads to the formation of a white precipitate that consists of SDS, lipids and proteins. In addition, the neutralization of the solution allows the renaturation of DNA. The large chromosomal DNA is captured in the precipitate, whereas the small plasmid DNA remains in solution. The precipitate and chromosomal DNA is removed by centrifugation.
Following centrifugation, the soluble plasmid DNA can be purified from the solution by various techniques. The most common is to precipitate the DNA with alcohol (ethanol or isopropanol) or high salt (ammonium acetate, lithium chloride, sodium chloride or sodium acetate). Another method is to bind the DNA to a solid support, such as glass fibers or silica. At high salt concentration and neutral or low pH, DNA molecules have a high binding affinity for these supports, allowing for the easy capture and subsequent elution of the DNA.