25.04.

2019

REPORT 5: MINIPREP PLASMID DNA

ISOLATION –BOILING METHOD

EDANUR ÇAKMAK
152204032

Group 8:
Aylin Yaşar
Şevval Çimyapan
Züleyha Özdemir
Edanur Çakmak
MINIPREP PLASMID DNA ISOLATION WITH THE BOILING METHOD

Purpose

The purpose of this experiment was to obtain plasmid DNA from recombinant cells. The obtained
plasmid DNAs was cut with restriction enzymes. The fragments will be present with agarose gel
electrophoresis. All in all, the suitability of the cloning will be determined.

Background Information
This Miniprep method is extremely fast, the quality of the DNA produced is lower than
the quality of alkaline lysis miniprep. In the alkaline lysis miniprep method, lysozyme is
used to hydrolyze large cross-linked proteins responsible for providing the strength of the
bacterial cell wall. The cells are then boiled to denature the proteins and disrupt the cell
walls. Plasmid DNA is then precipitated with alcohol. The yield and quality of plasmid
DNA is highly dependent on the type of culture medium used. Most plasmid purification
is optimized with cultures grown in standard Luria Bertani (LB) medium (1).

To find optimal culture conditions, the culture media and incubation times should be
individually optimized for each host strain / plasmid structure combination. Most
plasmids carry a marker gene for a specific antibiotic resistance. By supporting the
growth medium with the preferred antibiotic, only the cells containing the plasmid of
interest will multiply. Adding antibiotics to the desired concentration will help maximize
plasmid yields. Adding too much antibiotic can inhibit growth and little can cause a
mixed population of bacteria to grow with and without the plasmid (2).

Plasmids are circular double-stranded DNA molecules different from chromosomal DNA
of cells. The structure and description of a bacterial cell is guided by genetic materials
found in DNA. Some of the plasmids are not essential for survival of the host bacteria.
Although not required, the plasmids are encoded in bacterial chromosomal DNA in
unspecific rooms according to bacterial genetic diversity and plasticity. Plasmids, the
host, are there while living in environments that are lethal or restrictive for growth
outdoors. Antibiotic manufacturer and protein expression. Antibiotic resistance genes are
encoded on the plasmid when some bacteria do not remain in an antibiotic-containing
environment, providing bacteria with a competitive advantage over antibiotic-sensitive
species. As a tool, the plasmids can be modified to express the protein of interest (3).
Figure 1: You can see the boiling method principle.

Plasmids offer services based on an invaluable model of DNA replication, separation,

conjugation and evolution. Plasmids were a tool in gene cloning and were very important
to modern recombinant DNA technology as a tool for gene expression.
Isolation of plasmid DNA from bacteria is an important technique in molecular biology
and is an important step in the method of cloning, DNA sequencing, transfection and gene
therapy. These manipulations require the isolation of high purity plasmid DNA. Purified
plasmid DNA, digestion, cloning, PCR, transfection, in vitro translation, staining and
sequencing all molecular biology in the field for immediate use (4).
 Materials
Solutions Required
LB medium containing ampicillin
STET solution
Lysozyme (10 mg/ml)
Ammonium acetate (5 M)
Isopropanol, cold

Preparing of Solutions
STET solution

• 8 % ( w/v ) Sucrose (The sucrose prevents explosion of the cell completely. If the
cells explosion, chromosomal DNA releases and the purity of plasmid DNA
decreases.)

• 5% ( w/v ) Triton X-100 (The Triton X-100 is non-ionic detergent and separates cell
wall gently. Thus, the chromosomal DNA fragments occur lesser than ionic-detergent.)

• 50 mM EDTA, pH 8.0

• 50 mM Tris.Cl, pH 8.0

Lysozyme ( 10 mg/ml )

• 10 mg lysozyme(The lysozyme enzyme provides cell lysis.)

• 1 ml 10 mM Tris.Cl, pH 8.0

Ammonium acetate ( 5 M )

• 38.5 g Ammonium acetate (The cut chromosamal DNA is bound other cellular
components with ammonium acetate.)

• Add 𝑑𝐻2O to 100 ml

Procedure

➢ 10 ml sterile LB+amp medium was inoculated with a single white colony from the
transformation plate.
➢ İt was incubated at 37 °C with shaking at 200 rpm overnight to obtain a saturated culture.
➢ The cells were pelleted by centrifugation at 4000 rpm for 20 minutes at 4 oC and discarded
supernatant.
➢ The bacterial pellet was resuspended in 400 μl STET solution. This was transferred to a 1,5 ml
eppendorf tube.
➢ 25 μl (10 mg/ml) lysozyme solution was added and vortex.
➢ The tube was placed in a boiling water bath (100 oC) for 45 seconds. .
➢ It was spun in microcentrifuge at 13.000 rpm for 15 minutes.
➢ The pellet was taken by using a sterile toothpick.
➢ 200 μl 5 M NH4Ac and 800 μl cold isopropanol were mixed with vortex.
➢ It was placed at -20 °C for 30 minutes.
➢ This solution was spun microcentrifuge at maximum speed for10 minutes and pour off
supernatant.
➢ The DNA pellet was washed with 100 μl 70 % ethanol.
➢ It was centrifuged in the microfuge at 13.000 rpm for 5 min.
➢ The ethanol was removed and dried the pellet in a centrifugal evaporator for 10-20 minutes.
➢ The pellet was resuspended in 50 μl sterile dH2O and stored at -20 °C.
➢ 5 l of the resuspended DNA was used for a restriction digest.

RESULTS
 DISCUSSION

This method for screening colonies involves performing a restriction digestion followed by a plasmid miniprep.
Well isolated colonies are harvested from a plate and transferred to the culture medium containing the
appropriate antibiotic for selection. LB medium supplemented with antibiotics for Miniprep cultures ensures
that bacteria do not increase the ability of the antibiotic to select for the plasmid. The plasmids are cut with
EcoRI and HindIII. Subsequently, fragments of agarose gel may be observed. According to the agarose gel,
various types of fragments are obtained.
The boiling lysis method of plasmid isolation is quick and is recommended for isolation of small plasmids.
Plasmids larger than 10 kb should be isolated by other methods. In this method, a short heat treatment is applied
to the bacterial cells in the presence of lysozyme and triton X-100 in a boiling water bath. Because of its small
size, plasmid DNA exits the bacterial cell, whereas genomic DNA is trapped in the cell. High-speed
centrifugation then separates the plasmid DNA from the cell residues forming the pellet. The pellet was
removed and the plasmid DNA is recovered by the ethanol or isopropanol precipitation method.

REFERENCE
1-) http://mcblabprotocols.com/protocols/protocol-plasmid-isolation-boiling-method-miniprep/
(Access Date: 23.04.2019)
2-)https://link.springer.com/protocol/10.1385/0-89603-402-X:265 (Access Date: 23.04.2019)
3) https://www.ncbi.nlm.nih.gov/pubmed/6269464 (Access Date: 23.04.2019)
4-) https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1745-4581.2008.00113.x (Access Date: 23.04.2019)