2018

GEBZE TECHNICAL
UNIVERSITY
MOLECULAR GENETICS LABORATORY
TURKEY

EBRU AKHARMAN
142204026
17.05.2018
 MINIPREP PLASMID DNA ISOLATION WITH THE BOILING METHOD
AIM:

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic
recombination such as molecular cloning to bring together genetic material from multiple sources, creating
sequences that would not otherwise be found in the genome. Isolation of plasmid DNA is the discovery of the
DNA of that living organism after the destruction of the host cell membrane or living cell wall by chemical
means enzymes. The purpose of this experiment is to obtain plasmid DNA from recombinant cells. The
obtained plasmid DNAs will be cut with restriction enzymes. The fragments will be displayed with agarose gel
electrophoresis. Thus, the correctness of the cloning will be determined.

INTRODUCTION
Plasmids are circular double stranded DNA molecule that are distinct from the cells chromosomal DNA. The
structure and function of a bacterial cell is directed by the genetic material contained within the chromosomal
DNA. In some cases plasmids are generally not essential for the survival of the host bacterium. Although not
essential, plasmids contribute significantly to bacterial genetic diversity and plasticity by encoding functions
that might not be specified by the bacterial chromosomal DNA. Plasmids specify traits that allow the host to
persist in environments that would otherwise be either lethal or restrictive for growth. For example antibiotic
resistance and protein expression. Antibiotic resistance genes are often encoded by the plasmid, which allows
the bacteria to persist in an antibiotic containing environment, thereby providing the bacterium with a
competitive advantage over antibiotic-sensitive species. As a tool, plasmids can be modified to express the
protein of interest. Plasmids have served as invaluable model systems for the study of processes such as DNA
replication, segregation, conjugation, and evolution. Plasmids have been pivotal to modern recombinant DNA
technology as a tool in gene-cloning and as a vehicle for gene expression.

The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step
in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations
require the isolation of high purity plasmid DNA. The purified plasmid DNA can be used for immediate use in
all molecular biology procedures such as digestion with restriction enzymes, cloning, PCR, transfection, in vitro
translation, blotting and sequencing.

The boiling miniprep is recommended for preparing small amounts of plasmid DNA from a large number of
cultures. While this method is extremely quick, the quality of DNA produced is lower than that from the
alkaline lysis miniprep. In the alkaline lysis miniprep method, lysozyme is used to hydrolyze the extensive
crosslinked proteins that are responsible for giving the bacterial cell wall its strength. The cells are then boiled
to further denature the proteins and disrupt the cell walls. The plasmid DNA is then precipitated with alcohol.

Yield and quality of plasmid DNA highly depend on the type of culture media used. Most plasmid purification
are optimized with cultures grown in standard Luria Bertani (LB) medium. Also cares needs to taken, as
overgrowing a culture might lead to a higher percentage of dead or starving cells and the resulting plasmid
DNA might be partially degraded or contaminated with chromosomal DNA. To find the optimal culture
conditions, the culture medium and incubation times have to be optimized for each host strain / plasmid
construct combination individually. Most plasmids carry a marker gene for a specific antibiotic resistance. By
supplementing the growth medium with the antibiotic of choice, only cells containing the plasmid of interest
will propagate. Adding antibiotic to the required concentration will help to maximize plasmid yields. Adding
too much antibiotic can inhibit growth and too little may cause a mixed population of bacteria to grow both
with and without the plasmid of interest.
 MATERIALS
Solutions Required

 LB medium containing ampicillin

 STET solution
 Lysozyme ( 10 mg / ml )
 Ammonium acetate ( 5 0M )
 Isopropanol, cold

Prepairing The 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 𝑑𝐻2 O to 100 ml

METHOD
1. Inoculate 10 ml sterile LB+amp medium with a single white colony from the transformation plate.
2. Incubate at 37 °C with shaking at 200 rpm overnight to obtain a saturated culture.
3. Pellet the cells by centrifugation at 4000 rpm for 20 minutes at 4 °C and discard supernatant
4. Resuspend the bacterial pellet in 400 μl STET solution and transfer to a 1,5 ml eppendorf tube.
5. Add 25 μl (10 mg/ml) lysozyme solution. Vortex to achieve complete suspension. (Be sure cells are
completely resuspended in order to maximize the number of cells exposed to the lysozyme and consequently the
yield of plasmid DNA.)
6. Place tube in a boiling water bath (100 °C) for 45 seconds. (Heat and detergents cause the weakened cell walls
to break, releasing pasmid DNA and RNA, but not the larger bacterial chromosome which remains attached to or
trapped inside the lysed cells.)
7. Spin in microcentrifuge at 13.000 rpm for 15 minutes. (The pellet, which should be fairly gummy, contains
bacterial debris as well as chromosomal DNA. The supernatant contains plasmid DNA and RNA. )
8. Take the pellet by using a sterile toothpick.
9. Mix with 200 μl 5 M 𝑁𝐻4 𝐴𝑐 and 1 ml cold isopropanol and vortex.
10. Place at -20 °C for 30 minutes. (The cold isopropanol precipitates the plasmid DNA and cellular RNA.
Considerably shorter incubation periods (e.g. 2 to 5 min) may be sufficient for precipitation.)
11. Spin in microcentrifuge at maximum speed for10 minutes and pour off supernatant.
12. Wash the DNA pellet with 100 μl 70 % ethanol.
13. Centrifuge in the microfuge at 13.000 rpm for 5 min.
14. Remove the ethanol and dry the pellet in a centrifugal evaporator for 10-20 minutes.
15. Resuspend the pellet in 50 μl sterile 𝑑𝐻2 𝑂 and store at -20 °C.
16. Use 5 μl of the resuspended DNA for a restriction digest. (1 μl EcoRI and 1 μl HindIII are used for
restriction digestion.)
 RESULTS

Image 1: Agarose Gel Image 2: DNA Marker

The size of cloning vector is 2,7 kbp and size of cloning gene is 3,5 kbp. Thus the size of recombinant DNA is
6,2 kbp. According to the agarose gel and DNA marker, There are 3 bants in the holes of 1, 2, 3, 5, 6, 8, 9, 12,
13, 15. The size of forward bant is 2,7 kbp, the size of second bant is 3,5 kbp and the size of last bant is 6,2
kbp.

There is one bant in fourth hole and the size of this bant is 6,2 kbp. There is one bant in the holes of 16, 17,
18, 19, 20 and the size of bants is 2,7 kbp. There are two bants in the holes of 10, 11, 14 and the size of
forward bant is 2,7 kbp and the size of last bant is 3,5 kbp. There is not bant in the seventh hole.
 DISCUSSION
The classic method for screening colonies involves performing a plasmid miniprep followed by restriction
digestion. Well-isolated colonies are picked from a plate and transferred to culture medium containing the
appropriate antibiotic for selection. LB media supplemented with antibiotics for miniprep cultures to insure
that the bacteria do not outgrow the ability of the antibiotic to select for the plasmid. The plasmids are cut
with EcoRI and HindIII. Then, the fragments can be observed in agarose gel. According to agarose gel, several
type fragments are obtained. In this experiment, several problems can be. The cells can not be competent.
Competent cells may exhibit lower transformation efficiencies 5–6 weeks after preparation. Our cells waited
longly time. To verify that bacteria are competent, perform a test transformation using a known amount of a
standard supercoiled plasmid. The ligation can be unsuccessful. If ligation was successful, the banding pattern
of the ligation products should be different from that of the unligated sample. There are three and one band
in the agarose gel but only two bands must occur. There are several problems for high background. The vector
DNA can be unsuccessful dephosphorylation. The correct antibiotic can be lack in plates or the antibiotic is
inactive.

If we look to the holes of 1, 2, 3, 5, 6, 8, 9, 12, 13,15, three bands can seen. These three bands have different
sizes. The size of forward band is 2,7 kbp and this band is vector DNA. The size of middle band is 3,5 kbp and
this band is insert DNA. The size of last is 6,2 and this band is vector+insert DNAs. In this situation, we say that,
the restriction digestion are not enough in the holes of 1, 2, 3, 5, 6, 8, 9, 12, 13,15 but these DNAs are
recombinant DNAs. If restriction digestion can be successful, only two bands are observed on the agarose gel.

There is one band in the fourth hole. The size of this fragment is 6,2 kbp. Thus, we say that, The DNA in the
fourth hole is recombinant DNA but this DNA was not cut restriction enzymes. If restriction digestion can be
successful, only two bands are observed on the agarose gel.

There are two band in the 10th, 11th, 14th holes. The size of forward fragment is 2,7 kbp. The size of last
fragment is 3,5 kbp. Thus, we say that, these DNAs are recombinant DNAs and the restriction digestion are
correct.

There are one bant in the 16, 17, 18, 19, 20 holes and the size of the fragments are 2,7 kbp. We say that, these
fragments are not recombinant DNA and have only vector DNA.

RESOURCES
https://www.promega.com/-/media/files/resources/product-guides/subcloning-
notebook/screening_recombinants_row.pdf?la=en

https://books.google.com.tr/books?id=r6QC0hTwsrwC&pg=PA79&lpg=PA79&dq=miniprep+boiling+method&
source=bl&ots=zzVajfFSly&sig=RTiNQW3WuXJ9g73SVazUJigDCAI&hl=tr&sa=X&ved=0ahUKEwjvo-
OT2IPbAhWGB5oKHb0oA7g4HhDoAQg3MAI#v=onepage&q=miniprep%20boiling%20method&f=false

https://currentprotocols.onlinelibrary.wiley.com/doi/pdf/10.1002/0471142727.mb0106s15

https://www.labome.com/method/DNA-Extraction-and-Purification.html

https://worldwide.promega.com/resources/product-guides-and-selectors/protocols-and-applications-
guide/dna-purification/

http://home.sandiego.edu/~josephprovost/Plasmid%20Prep%20Lab.pdf