Practical Molecular Biology Lab. 03 November 8, 2020

Practical Molecular Biology

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Objectives (Purposes):

Explain the process of isolating plasmid DNA (DNA extraction procedure from prokaryotic Cell).

How many types of purification methods for Bacterial Plasmid Isolation?

What are the roles of different chemicals that are used during Isolation-Purification of Plasmid

DNA?

Plasmid Background:

A plasmid is an extrachromosomal DNA molecule separate from the

chromosomal DNA which is capable of replicating and transmitting independently of

the nuclear DNA. It is Circular, supercoiled and double-stranded DNA molecules

present in most species but not all strains of bacteria. Plasmid size varies from 1 to over

250 kbp and ranging from 0.2 to 4 percent of bacterial chromosome. The number of

identical plasmids within a single cell can be zero, one, or even hundreds under some

circumstances. Plasmids are much smaller than bacterial chromosomes, the largest

plasmids being only 8% of the size of E. coli chromosome.

Purification of Total Cellular DNA from Prokaryotic Cells

The DNA obtained from prokaryotes is the following three types:

1) Total Cell DNA.

2) Plasmid DNA.

3) Phage DNA.

Preparation of total cell DNA from bacteria consists of the following four steps:

1. Multiplication of the bacterial cells (Plasmid Amplification).

2. Rupture/lysis of cells to obtain cell extract.

3. Purification of DNA.

4. Concentration of DNA.

Purification of Plasmid DNA (Principles for Plasmid Isolation)

Plasmids are most commonly used vectors in gene cloning work and plasmid

DNA is isolated from cells of appropriate bacterial strains, which are grown in a suitable

liquid medium (for increasing copies of DNA plasmid, called Plasmid amplification).

Isolation is naturally a very routine requirement. There are many methods developed for

isolation and purification of plasmid but none of them are problem free. Most critical

stage in the process is lysis of the cell which is just sufficient for plasmid DNA to escape

but without much contamination by chromosomal DNA. If lysis is done in gentle

manner high molecular weight chromosomal DNA should remain along with cell debris

when high speed centrifugation is carried out. Clear lysate then contains plasmid.

Electron micrograph of DNA

from a lysed E. coli cell

Plasmid DNA can be isolated from bacteria in a simple way:

1. Plasmid- containing bacteria are opened by lysozyme, detergent treatment and

the resulting translucent solution called a cell lysate is centrifuged.

2. The bacterial chromosome complex, which contains protein and RNA, is

very large and compact and sediment to the bottom of the centrifuge tube; the

smaller plasmid DNA remain in the clear supernatant, which is called a clear

lysate.

3. Some Chromosomal DNA is usually present in the clear lysate, but since most

of the plasmid DNA is covalently circular, this contaminating material can be

easily removed by the following procedure:

a) CsCl2 (Caesium Chloride)-EtBr (Ethidium Bromide) density Gradient

Centrifugation.

b) Alkaline-SDS Lysis Denaturation.

There are several methods to isolate plasmid DNA from bacteria:

I. Mini Plasmid prep Method:

Can be used to quickly find out whether the plasmid is correct in any

of several bacterial clones. The yield is a small amount of impure plasmid

DNA 1 μg/ml of the culture), which is sufficient for analysis by restriction

digest and for some cloning techniques.

II. Maxi Plasmid prep / Bulk prep Method:

Much larger volumes of bacterial suspension are grown from which

a maxi-prep can be performed and it is used for isolation of mg quantities of

plasmid DNA. Essentially this is a scaled-up miniprep followed by additional

purification. This results in relatively large amounts (several micrograms) of

very pure plasmid DNA. It’s using in PCR techniques and Southern Blotting

Techniques……..etc.

Plasmid Applications:

The plasmids used in transformation typically have three important elements:

A cloning site (a place to insert foreign DNAs).

An origin of replication.

A selectable marker gene (e.g. resistance to ampicillin)

Practical Molecular Biology Lab. 03 November 8, 2020

Practical Molecular Biology

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Cells that contain the plasmid have been grown in Lurial-Bertani or Luria broth

(LB) storage medium or Nutrient Broth or Brain Heart Infusion Broth containing 20

mg/l ampicillin.

The protocol used to extract plasmid DNA from bacterial cell suspensions is based

on the alkaline lysis procedure. The cells are lysed under alkaline conditions, which

denature both nucleic acids and proteins, and when the solution is neutralized by the

addition of potassium acetate, chromosomal DNA and proteins precipitate because they

can't renature correctly (they are so large). Plasmids renature correctly and stay in

solution, effectively separating them from chromosomal DNA and proteins.

AddPrep Plasmid DNA Extraction Kit offers a simple, rapid and cost-

effective method for isolating plasmid DNA from bacterial cells. This kit is

designed for the preparation of up 20 µg of high-purity plasmid DNA from 1

~ 5 ml overnight E. coli culture in LB medium. Plasmid DNA purified with

the mini kit is immediately ready for use. Phenol extraction and ethanol

precipitation are not required a high-quality plasmid DNA are eluted in a small

volume of Elution Buffer.

Extraction Protocol

1. Growth of bacterial cultures in tubes or flasks and harvesting: Harvest the

bacterial cells from 1 ~ 3ml recombinant E. coli culture by centrifugation of

8,000 rpm in a conventional, table-top microcentrifuge for 3 min at room

temperature.

2. Add 250 µl of Resuspension Solution to the collected cells and completely

re-suspend by vortexing or pipetting.

3. Add 250 µl of Lysis Solution and mix by inverting the tube 3~5 times by

inverting, gently: Vortexing may cause shearing of genomic DNA. Do not

vortex.

4. Add 350 µl of Neutralization Solution and immediately mix by inverting

the tube 3~5 times, gently: Genomic DNA and cell debris will be formed and

insoluble complex. Do not vortex.

5. Centrifugation of the tube at 13,000 rpm, 4 ~ 25℃ for 10 min in micro-

centrifuge: A compact white pellet will be appeared at the bottom of the tube.

6. Transfer the supernatant (cleared lysate) to the spin column with a

collection tube and centrifuge at 13,000 rpm for 1 min: Pour off the flow-

through and assemble the spin column with the 2.0 ml collection tube.

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7. (Optional) Add 500 µl of Optional Solution and wait 5 min and centrifuge

at 13,000 rpm for 1 min: Pour off the flow-through and assemble the spin

column with the 2.0 ml collection tube.

8. Add 700 µl of Washing Solution to the spin column with a collection tube

and centrifuge at 13,000 rpm for 1 min: Pour off the flowthrough and

assemble the spin column with the 2.0 ml collection tube.

9. Dry the spin column by additional centrifugation at 13,000 rpm for 1 min

to remove the residual ethanol in the spin column.

10. Transfer the spin column to the new 1.5 ml microcentrifuge tube (Not

provided).

11. Add 50 ~ 100 µl of Elution Solution to the spin column with a micro-

centrifuge tube, and let stand for at least 1 min.

12. Elute the plasmid DNA by centrifugation at 13,000 rpm for 1 min.

Elution Buffer (Tris EDTA TE Buffer) is commonly used to redissolve DNA

because it contains EDTA. The EDTA will chelate magnesium ions, which are a

cofactor for most nucleases (enzymes that degrade nucleic acids). If your DNA prep

becomes contaminated with a nuclease, then the nuclease will be inactivated by the

fact that the magnesium cofactor is unavailable in the solution.