Molecular Biology Laboratory Practice Manual I

Laboratory Practice Manual for Introduction to Molecular Biology I
Laboratory Practice Manual

Introduction to Molecular Biology I
Authors
Ricardo Carreño López, Fabiola Avelino
Flowers, María del Rocío Bustillos Cristales,
Edith Chávez Bravo, Rocío Pérez y Terrón,
Lorena Milflores Flores
Bachelor's degree in Biotechnology

Benemérita Autonomous University of
Puebla
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Authors
Ricardo Carreño López

Doctor in Microbiological Sciences, Research Professor, Microbiological Sciences
Research Center of the Institute of Sciences of the Benemérita Universidad
Autónoma de Puebla. Puebla, Mexico. Email: ricardo.carreno@correo.buap.mx
Fabiola Avelino Flores

Doctor in Environmental Sciences, in the Area of Environment and Health. Research
Professor, Microbiological Sciences Research Center of the Institute of Sciences of
the Benemérita Universidad Autónoma de Puebla. Puebla, Mexico. Email:
avelinofloresf@yahoo.com. Microbial Pathogenicity Laboratory.
María del Rocío Bustillos Cristales

PhD candidate in Microbiology. Research Professor, Microbiological Sciences
Research Center of the Institute of Sciences of the Benemérita Universidad
Autónoma de Puebla. Puebla, Mexico. Email: mrbust@yahoo.com
Edith Chavez Bravo

Doctor in Environmental Sciences, in the Area of Environment and Health. Research
Professor, Microbiological Sciences Research Center of the Institute of Sciences of
the Benemérita Universidad Autónoma de Puebla. Puebla, Mexico. Email:
echb_02@yahoo.com.mx. Microbial Pathogenicity Laboratory.
Rocío Pérez and Terrón

Doctor in... Research Professor, School of Biology of the Benemérita Universidad
Autónoma de Puebla. Puebla, Mexico. Email: rocperez33@hotmail.com
Lorena Milflores Flores

Doctor in Chemical Sciences, Research Professor. School of Biology of the Benemérita Universidad
Autónoma de Puebla, Puebla, Mexico. Email: lorenamilflo@yahoo.com.mx. Molecular Biology and
Microbiology Laboratory.
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Index
General rules for the use of the Biology laboratory Molecular.................................................7

Genomic DNA Extraction: Traditional Method and Kit...........................................................10
Aim .......................................................................................................................................10
Introduction ..........................................................................................................................10
Expected results.....................................................................................................................15
Questionnaire ........................................................................................................................15
Preparation of solutions and reagents....................................................................................16
Bibliography..........................................................................................................................16
Extraction of plasmid DNA by traditional method...................................................................17
Aim........................................................................................................................................17
Introduction...........................................................................................................................17
Expected results.....................................................................................................................19
Questionnaire ........................................................................................................................19
Bibliography..........................................................................................................................21
Purification of plasmid DNA by silica......................................................................................22
Aim .......................................................................................................................................22
Introduction...........................................................................................................................22
Protocol.................................................................................................................................23
Results to document..............................................................................................................24
Questionnaire.........................................................................................................................24
Preparation of Solutions and reagents...................................................................................25
Bibliography..........................................................................................................................26
TO..........................................................................................................................................26
Purification of chromosomal DNA and plasmid DNA.............................................................28
Aim .......................................................................................................................................28
Introduction...........................................................................................................................28
Bibliography..........................................................................................................................35
DNA quantification in agarose gels..........................................................................................35
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Aim........................................................................................................................................35
Introduction ..........................................................................................................................36
Protocol ................................................................................................................................37
Questionnaire ........................................................................................................................39
Note :.....................................................................................................................................40
Bibliography..........................................................................................................................40
DNA quantification by spectrophotometry...............................................................................41
Aim .......................................................................................................................................41
Introduction ..........................................................................................................................41
Protocol ................................................................................................................................42
Questionnaire.........................................................................................................................43
TO..........................................................................................................................................43
c.............................................................................................................................................43
Bibliography..........................................................................................................................44
Extraction by bacterial RNA kit................................................................................................45
Aim .......................................................................................................................................45
Introduction ..........................................................................................................................45
Protocol.................................................................................................................................46
Questionnaire.........................................................................................................................48
Bibliography..........................................................................................................................48
Conclusion of the manual.........................................................................................................49
General Bibliography................................................................................................................50
General objective
That the student learns to perform and compare the different methodologies for purification
and analysis of nucleic acids.
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Summary
Molecular biology as a scientific discipline whose objective is the study of the processes that
develop in living beings from a molecular point of view. Trying to explain the phenomena of life from
their macromolecular properties. Especially through the study of nucleic acids and proteins.
By studying the molecules that make up living cells, Molecular Biology borders on the
methodologies used by other scientific disciplines that address similar topics such as genetics, genetic
engineering and biochemistry, among others.
For the professional who will develop in the field of biotechnology taking advantage of the
qualities of microorganisms, eukaryotic cells and active subcellular structures, it is of utmost
importance to learn to manipulate and analyze nucleic acids.
The methods of manipulation and analysis of nucleic acids are of special importance when we
want to know the composition, sequence, size and mode of action of a gene to follow the process of
transmission of this genetic information to RNA and subsequently to a possible peptide, protein and /or
enzyme.
This manual is intended to give the student basic information and procedures for the
manipulation of nucleic acids. Different DNA purification methodologies are addressed so that the
student can judge the advantages and disadvantages of each of them, as well as the qualitative and
quantitative detection methods of DNA and RNA.
It is a basic work that in its sections attempts to complement the theoretical information that
the student has received with methodologies for purification and analysis of nucleic acids.
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General rules for the use of the Biology laboratory

Molecular
For the development of laboratory practices, it is advisable to take into account some elementary rules
that the student must follow:
1. Before starting the laboratory practice, you must carefully read the basic laboratory rules of
Molecular Biology, as well as the respective practice to be developed, in order to have a clear idea
of the objective, foundation and methodology to follow.
2. Personal accidents, such as spilled reagents, cuts, burns or others, must be reported immediately
to the Professor-Instructor.
3. Order and cleanliness are essential before, during and after carrying out the laboratory practice.
Each group or section of students will be responsible for their work area and their material.
4. No food or drinks may be consumed inside the laboratory, nor will playing or making jokes be
avoided during the development of the practice.
5. Flammable products (gases, alcohols, ether, chloroform, etc.) must be kept away from flames, for
example from lighters. If gas burners are used, great care must be taken to close the stopcocks
when turning off the flame.
6. Before using a reagent, you must look at the label to make sure it is the one you need, the way it is
handled, and the possible risks to your health.
7. All materials and equipment, especially delicate devices, such as electrophoresis chambers, must
be handled with care, avoiding impacts or forcing their opening and closing mechanisms.
Special attention must be paid when using the electrophoretic chamber. The minimum muscle
contraction threshold occurs at 9 mA, which may affect the respiratory muscles.
The dangerous current threshold occurs at 80 mA in alternating current, which can cause
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ventricular fibrillation with possible temporary cardiac and respiratory paralysis. In
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In general terms, a current, either direct or alternating, of 100 mA is dangerously lethal.

When using pressure cookers and microwave ovens, extreme care should be taken to avoid
sealing jars when introducing them to these devices and to avoid introducing metals into the
microwave oven.
8. All waste materials that have come into contact with microorganisms (such as pipettes, Petri
dishes, tubes, micropipette tips, etc.) must be placed in autoclave bags prepared for this purpose
and subsequently sterilized. The glass Pasteur pipettes, coverslips and used slides will be placed
in a special glass container that will be indicated by the teacher before starting the practice.
9. Always wear a “coat” in the laboratory, as well as gloves, face masks, glasses, etc. when the
instructor instructor indicates it or it is indicated in the laboratory practice that will be carried out.
Do not touch solvents with your bare hand such as acetone, methanol, etc.
If you wear contact lenses, the risk is greater for your eyes with gases because they can be
occluded behind the lenses.
10. Cultures of microorganisms or chemicals should never be stolen from the laboratory.
11. Wash your hands with soap or disinfectant if necessary, frequently during the practice and always
before leaving the laboratory.
12. The inoculated culture media must be placed in the culture chambers with their respective
identification, e.g.: name of the microorganism, name of the student, nature of the medium, date,
etc.
13. Do not throw chemical waste into the drain, you must inform your Professor-instructor how to
dispose of it.
14. Micropipettes should be used according to the teacher's instructions. If you are not sure how to use
them, you should ask the instructor to avoid decalibration. Remember that micropipettes, despite
being a small instrument, are highly accurate and expensive.
15. The cells for the spectrophotometer, and the coverslips and slides must be held by the edges to
prevent them from becoming greasy, and their manipulation must be firm and delicate to avoid
damage.
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16. Test tubes containing culture media or cultures of microorganisms should never be opened in a
vertical position, but rather as horizontally as possible (inclined) and to remove the stopper they
should be kept inclined with one hand and opened with the other, which will hold the turn the
handle. Once opened, the hole is flamed for a few seconds, repeating this operation once the
sowing is done (the plug should never be left on the table).
17. Before using the handles with platinum threads that are used for sowing, they must be flamed red
in a vertical position under the action of the flame; The handle must also be flamed. Before sowing,
wait a few seconds for them to cool, and they may also cool on the edge of the Petri dish
containing the culture medium. Immediately after using them, they should be flamed again to
eliminate any microorganisms that are still in it.
18. The student must be methodical in carrying out the laboratory practice and write down the results
immediately after obtaining them.
19. If you have doubts or questions, you should contact the Professor-Instructor, never with your fellow
student.
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Genomic DNA Extraction: Traditional Method and Kit
Aim .
Extract genomic DNA from a Gram-negative bacteria using a traditional method and genomic
DNA from blood with a commercial kit.
Introduction .
The extraction and purification of nucleic acids is the first step in most molecular biology
studies. To manipulate or amplify DNA, it is necessary to eliminate other cellular components that can
interfere with experiments, such as proteins, RNA, and lipids without damaging the DNA. There are
several methods for the extraction and purification of nucleic acids, which are selected according to:
• The type of nucleic acid to be extracted (genomic DNA, plasmid DNA, total RNA, messenger
RNA).
• The organism from which it will be extracted (animal cells, plants, yeasts, bacteria, viruses)
• The extraction material (whole organs, tissues, cell cultures, blood, environmental samples)
• The desired results (yields, purity, purification time)
• The subsequent application (amplification, cloning, expression, reverse transcription).
The extraction of nucleic acids from biological materials requires homogenization of the
tissues, where appropriate, the lysing of the cells and the inactivation of cellular nucleases that could
digest the DNA. This ensures that the amount of intact DNA obtained is the maximum.
Homogenization and cell lysis must be strong enough to disrupt tissue, cell wall, and membranes, but
be gentle to preserve nucleic acids. The most common processes are:
• Mechanical disruption, such as hypotonic disruption, or freeze-thaw.

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• Chemical treatment, such as lysis with detergents or chaotropic agents.
• Enzymatic digestion with proteases, lysozyme or mutanolysin.
Cell disruption and inactivation of intracellular nucleases must be combined. A solution can be
used with detergents to solubilize cell membranes and strong chaotropic salts to inactivate nucleases.
DNA and other cellular components such as lipids, sugars and proteins are dissolved in the lysis
solution. DNA has a negative charge due to the phosphate groups in its structure, and this electrical
charge is what makes this molecule soluble.
After extraction, the DNA must be purified and this is frequently achieved with combined
methods such as:
• Extraction/precipitation using solvents to remove contaminants, such as combining phenol and

chloroform to remove proteins; by using high concentrations of salt or changes in pH to
precipitate proteins (selective precipitation); or the precipitation of nucleic acids with
isopropanol or ethanol, since DNA is insoluble in high concentrations of salt and alcohol. The
precipitated DNA forms thin white strands, while the rest of the substances remain dissolved.
• Chromatography which can be carried out by gel filtration, separating the molecules by their
molecular size; by ion exchange, separating and concentrating the molecules by electrostatic
interactions with the column matrix; by adsorption, using silica membranes in the presence of
high concentrations of certain salts and subsequent elution with water or a buffer; by affinity,
binding the nucleic acids to a particular matrix, and subsequently a molecule is added that
competes for the ligand, leaving the nucleic acids free.
• Centrifugation, which is an important purification method frequently used in combination with

other methods, such as gel filtration and adsorption chromatography.
• Agarose gel electrophoresis, frequently used to determine the size and integrity of extracted
DNA.
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Requirements.
Equipment Material Reagents
Extraction hood 15 mL falcon tubes Medium LB
Vortex Sterile double-distilled water or

1.5 mL microcentrifuge tubes, sterile
Milli-Q TM
Incubator with Micropipette from 100 to 1,000 uL and Phenol balanced with Tris
sterile disposable tips
orbital shaking at 37 °C
Microcentrifuge Micropipette from 10 to 100 3M Sodium Acetate with pH

uL and sterile disposable tips
5.2
Extraction hood Strain of E. coli K-12 absolute ethanol
Freezer at -20°C Blood 1 mL Chloroform
Water bath Zymo Research Quick-gDNA TE Buffer pH 8.0

MiniPrep Kit
Protocol .
Extraction of genomic DNA from an Escherichia coli strain.
1. Propagate a culture of E. coli in LB medium (5 mL) overnight (18-24 hours)

at 37°C with constant stirring of 200 rpm.
2. Place 1 mL of the culture in a 1.5 mL polypropylene tube, centrifuge the tube at

4,000 rpm for 5 minutes and decant the supernatant.
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3. Add 250 µL of water sterile deionized to cell pack and shake

vigorously using a vortex until resuspension of the cells is achieved (approximately 30 s).
4. Add 250 µL of phenol (equilibrated with 1M Tris pH 8.0) and shake vigorously for 30 s using a
vortex.
5. Add 250 µL of chloroform and shake vigorously using a vortex for

30s.
6. Centrifuge at 9,000 rpm for 5 minutes.
7. Recover the aqueous phase (upper phase, approx. 225 µL) and place it in another 1.5 mL tube.
On many occasions, a white precipitate occurs at the interface; this precipitate should not be
recovered, since they are proteins that can contaminate the sample and inhibit subsequent
reactions.
Aqueous
phase
Interface
8. Add 225 µL of chloroform and shake vigorously for 30 s using a vortex.
9. Centrifuge at 9,000 rpm for 5 minutes.

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10. Recover the aqueous phase (upper phase, approx. 200 µL) and place it in another 1.5 mL
tube, taking the same precautions as in step VII.
11. Add 20 µL of Sodium Acetate (3M) to the aqueous phase, vortex for 20 s and add 440 µL of
cold Absolute ethanol to the aqueous phase.
12. Centrifuge at 12,000 rpm for 30 minutes. It is important to always place the tubes in the same
orientation, to know which side of the tube the precipitated DNA is on.
13. Remove the supernatant and let it dry at 37ºC.
14. Resuspend in 100 uL of TE or PCR grade water and store at -20 ºC. To use it in subsequent
analyses, the degree of purity must be quantified and determined.
Extraction of genomic DNA from blood with the Quick-gDNA MiniPrep kit.
1. Add 400 μl of Lysis Buffer to 100 μl of blood in a 1.5 mL microtube (4:1). Mix thoroughly with
mechanical shaking for 6-10 seconds, incubate at room temperature for 5-10 minutes.
2. Transfer the mixture to a Zymo-Spin column (attached to a collection tube) and centrifuge at
11,000 rpm for one minute. Discard the collection tube along with the liquid content.
3. Transfer the column to a new collection tube, add 200 μL of the Pre-Wash Buffer and
centrifuge again at 11,000 rpm for one minute. Discard the collection tube along with the liquid
content.
4. Add 500 μL of Wash Buffer to the column and centrifuge at 11,000 rpm for one minute.
Discard the collection tube along with the liquid content.
5. Transfer the column to a clean, sterile microcentrifuge tube. Add > 50 μL of DNA Elution Buffer
or sterile water. Incubate for 5 minutes at room temperature. Centrifuge at 12,000 rpm for 45
seconds to elute the DNA. The DNA obtained can be used immediately or frozen at -20°C.
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Blood Sample + Genomic Lysis

Buffer
Centrifuge
Wash Elute
Ultrapure DNA for: PCR

Endonuclease digestion
Sequencing
Expected results.
In both cases you will obtain a transparent resuspension that you will store at -20°C so that it
runs on the agarose gels at the time indicated by your instructor. Photodocument the results obtained
and discuss the differences observed in the gel.
Questionnaire .
1. What DNA extraction processes were used in each case?

2. What precautions should be taken when handling phenol?
3. Will it be the same to obtain DNA from E. coli and human blood? Support your
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answer.
4. What is the function of the column in the kit DNA extraction method?
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5. What would be the applications of these techniques in Biotechnology?
Preparation of solutions and reagents.
1. Phenol balanced with 1 M Tris pH 8.0

2. Sodium acetate 3M pH 5.2
3. TE Buffer pH 8.0: 10 mM Tris-HCl, 1 mM EDTA. Sterilize and keep
room temperature.
Bibliography.
Green MR and Sambrook J. (2012). Molecular cloning: A Laboratory Manual (4th edition).
Woodbury, New York: Cold Spring Harbor Laboratory Press.
Madigan MT, Martinko JM, Bender KS, Buckley D. H., , Stahl D. TO. and Brock T. (2015).
Brock Biology of microorganisms (14th edition). Madrid, Spain: Pearson.
ZymoResearch. (2014). Quick-gDNA MiniPrep Kit Instruction Manual

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Extraction of plasmid DNA by traditional method
Aim.
Isolate plasmid DNA from prokaryotic cells using a traditional method.
Introduction.
Plasmids are small circular DNA molecules (between one and several hundred kilobases) that
replicate autonomously and independently of the cell's chromosome. The vast majority of plasmids are
double-stranded DNA (3). They are very common in bacteria and some of them, the smallest ones,
exist in bacterial cells in high numbers; they are characterized by carrying extra genetic information
that is essential for the adaptation of bacteria (1).
Most plasmids are characterized by:
• Have an autonomous replication origin.
• Having a gene that gives the bacteria resistance to antibiotics or heavy metals, serves to
perform some metabolic function, and/or codes for some virulence factor.
• Possess a multiple cloning site in which unique targets for several restriction enzymes are
found and which allows the introduction of foreign DNA (3).
There are several procedures for the purification of plasmid DNA, although all include the
following three steps: (i) Growth of bacteria in a medium selective for those that carry the plasmid. (ii)
Lysis of the bacteria for the release of the plasmid. (iii) Purification of plasmid DNA. One of the most
used methods is the “alkaline lysis” method, due to its simplicity, low cost and reproducibility.
Alkaline lysis utilizes the differences in denaturation and renaturation properties between
plasmid DNA and chromosomal DNA. Alkalinization with NaOH in the presence of a strongly anionic
detergent (SDS) causes cell lysis, denaturation of chromosomal DNA and proteins, and release of
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plasmids. Plasmids are less affected by their small size and supercoiled structure. The neutralization
of the medium in the presence of a high concentration of salt (potassium acetate) causes the
precipitation of the proteins (due to the treatment with the detergent and the insolubility of the
potassium salt of the SDS) and that of the chromosomal DNA (due to random intrastrand
reassociations). ). The insoluble aggregates of proteins and chromosomal DNA are separated by
centrifugation from the plasmid DNA that remains in the supernatant and mostly retains its native
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structure (2).
Requirements :
Shaking incubator 15 mL falcon tubes LB Broth
Microcentrifuge 1.5 mL eppendorf tubes Birnie I Solution
Vortex Birnie II Solution

Multi-volume micropipettes
New, sterile blue, yellow and

Fridge Birnie III Solution
white tips
Vacuum concentrator Ice Cold absolute isopropanol
cold absolute ethanol
75% ethanol
Sterile double-distilled water or

Milli-Q TM
Protocol
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1. Centrifuge 1.5 mL of a 24-h bacterial culture at 8000 rpm for 2 minutes.
2. Aspirate the supernatant with the help of a sterile tip and resuspend the cell package in 100
uL of Birnie I solution. Incubate for 5 minutes at room temperature.
3. Add 200
uL of Birnie II solution and mix gently by inverting gently for 10 times (The solution
should become clear). Incubate for 5 minutes on ice.
4. Add 150
uL of Birnie III. Incubate for 15-30 minutes on ice.
5. Centrifuge at 12000 rpm for 10 minutes. Add 600

uL of isopropanol and let it rest
for 10 minutes.
6. Centrifuge at 14000 rpm for 10 minutes.
7. Remove the supernatant and wash the package with 1000

uL of cold 75% ethanol, mix by
inversion gently 7 to 10 times and centrifuge for 10 minutes at 14000 rpm.
8. Remove as much of the ethanol as possible and dry the tube in the vacuum concentrator for
approximately 5 minutes.
9. Resuspend the DNA in 50 uL of sterile double-distilled water and store at -20°C.
Expected results.
You will obtain a transparent resuspension that you will store at -20°C so that it runs on the
agarose gels at the time indicated by your instructor. Photodocument the results observed in the gel.
Questionnaire .
1. What is the purpose of neutralization in the “alkaline lysis” method?
2. Why does alkalinization in the presence of SDS affect plasmids less than chromosomal DNA?
3. What is the purpose of the ribonuclease A incubation step in plasmid DNA purification?
4. What uses can be given to plasmids in Biotechnology?

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Preparation of solutions and reagents
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1. LB medium: 10 g/L casein peptone, 5 g/L yeast extract, 10 g/L NaCl.

2. Birnie I: 25 mM Tris-HCl, 10 mM ethylenediamine tetracetic acid (EDTA) and RNase H or A,
pH 8.0.
3. Birnie II: 0.2 N NaOH and 1.0% sodium dodecyl sulfate (SDS)
4. Birnie III: 3 M Potassium Acetate (KOAc), pH 4.8
Bibliography
Balbas P. (2010). From Molecular Biology to Biotechnology (2nd. edic). Mexico: Trillas.
Green MR and Sambrook J. (2012). Molecular cloning: A Laboratory Manual (4th edition). Woodbury,
New York: Cold Spring Harbor Laboratory Press.
Madigan MT, Martinko JM, Bender KS, Buckley D. H.,, Stahl D. TO. and Brock T. (2015).
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Purification of plasmid DNA by silica.
Aim .
Purify plasmid DNA by using silica.
Introduction.
The isolation of plasmid DNA from bacteria is fundamental in molecular biology and is an
essential step in many procedures such as cloning, DNA sequencing, transfection and gene therapy.
These procedures where DNA is manipulated require the isolation of high purity plasmid DNA. There
are anion exchange columns for sale on the market, although widely used, they are relatively
expensive. A less expensive alternative method uses silica oxide as the DNA binding matrix. But it has
the disadvantage that the bacterial lipopolysaccharide (LPS) or endotoxin, are copurified and can
inhibit possible subsequent applications. In particular, the Lipopolysaccharide content can greatly
influence the transfection by influencing the efficiency. Furthermore, these methods usually require the
use of hazardous chemicals (e.g., guanidine hydrochloride), which act as chaotropic substances to
facilitate the binding of DNA to silica oxide. A slightly modified procedure is the following that is easy,
fast and relatively inexpensive.
Requirements:
Horizontal electrophoresis Polypropylene pipes Biology grade agarose

chamber 1.5mL Molecular
Microcentrifuge (14000 Yellow and blue tips Solution P1

rpm)
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Power supply 15 mL falcon tubes Solution P2
Incubator Escherichia coli (pTZ18R) Solution P3
Bacteriological loop NaI 6M solution
Plates Petri 90X15 Marker of Weight

disposable 1 KB DNA molecular
2-20 µL, 20-200 µL and 200-

Wash solution (New Wash)
1000 µL micropipettes
Sterile double-distilled water or

Milli-Q TM
LB* agar
Glycerol
Bromophenol blue
Ampicillin
Silica
Acetic acid
Protocol.
An Escherichia coli colony containing a plasmid of interest (e.g. pTZ18R) is

inoculated into a tube containing 5 mL of culture medium (LB) with the appropriate
antibiotic (Amp). Incubate overnight at 37°C at 160 rpm.
1. 1.5 mL of bacterial culture are transferred to a polypropylene tube.

“eppendorf” and centrifuged at 5000 xg for 5 minutes. The supernatant was C
Discard it in a suitable container to later sterilize it.
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~(
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2. The pellet (bacterial cells) free of culture medium is resuspended with 100 µL of P1
solution (with vortex or micropipette).
3. Add 200 µL of P2 lysis solution, invert the tube 4-6 times (do not use vortex), letting
it rest on ice for 5 minutes.
4. Add 150 µL of P3 neutralization solution, invert the tube 4-6 times (do not use
vortex), let it rest for 10 minutes on ice and centrifuge at maximum speed for 10
minutes.
5. Recover supernatant and add 800 µL of NaI binding solution, mix by inversion and
add 5 µL of silica suspension, mix by inversion and incubate at room temperature for
5-7 minutes.
6. Centrifuge at 14000 rpm for 30 s and remove all supernatant by decanting and
pipetting.
7. Add 800 µL of new wash and resuspend the silica by inversion, centrifuge at 14,000
rpm/30 s, repeat the procedure if a better quality plasmid is required.
8. Dry at 42°C for 2-5 minutes
9. Add 50 µL of water with RNase and incubate for 1 hour, centrifuge and recover the
supernatant.
Results to document.
1. Observe and document how the cellular package undergoes modifications when
adding the different solutions;
2. Observe and document the difference between the cell package and the DNA
obtained from it by the in silica methodology;
3. Document the result of electrophoretic analysis of the purified DNA;
4. Compare the plasmid DNA purification results with other methodologies carried out.
Questionnaire.
1. Explain why it is necessary to culture the bacteria Escherichia coli (pTZ18R) with N
the antibiotic Ampicillin;
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2. Describe the function of each solution in the DNA purification process using this
methodology.
3. Explain why it is necessary to completely remove the alcohol contained in the wash
solution in order to elute the DNA from the silica.
Preparation of Solutions and reagents.
P1 : 50 mM Tris/HCl (pH 8.0) P2 : 200 mM NaOH, 1% SDS
10mM EDTA
100 µg/mL RNase A
P3 : 3.0 M potassium acetate, pH 5.5
Preparation of silica suspension
Grind with mortar and pestle 2 new Pasteur pipettes
Resuspend in the same volume of water (Pisa)
Let rest 5 minutes
Take the upper phase and dry
Resuspend in the same volume of water (pisa)
Take 1 mL of the upper phase in an Eppendorf tube and centrifuge for 1 minute at
14000rpm.
Wash 6 times with the same volume of water (pisa)
Resuspend in water (step approx. 1ml)
Silika DNA Binding Solution
• NaI 6M
c NaI or KI 90 g
c
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.AND Sodium sulfite 1g

60 9
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CBP 100 mL water
Filter with paper and store in an opaque container away from light.
• Wash solution (New Wash)
100mM NaCl
1 mM EDTA
10 mM Tris-HCl, pH 7.5
50% Ethanol
Bibliography.
Bert Vogelstein and David Gillespiet. (1979). Preparative and analytical purification of DNA
from agarose. Proc. Natl. Academic Sci. USA 76(2): 615-619.
Debra A. Dederich, Geoffrey Okwuonu, Toni Garner, Amanda Denn, Angelica Sutton,
Michael Escotto, Ashley Martindale, Iliver Delgado, Donna M Muzny, Richard A
Gibbs and Michael L. Metzker. (2002). Glass bead purification of plasmid template
DNA for high throughput sequencing of mammalian genomes. Nucl Acds research
30(7): 1-5.
Etienne Joly. (1996). Purification of DNA Fragments from Agarose Gels Using Glass
Beads. Basic DNA and RNA Protocols. Methods Mol Biol. 58: 237-240.
Hamid Kheyrodin1 and Khosro Ghazvinian. (2012). DNA purification and isolation of
genomic DNA from bacterial species by plasmid purification system. African J
Agricultural Research 7(3): 433-442.
Karl-Heinz Esser1, Dr. Wolfram H. Marx2 and Prof. Dr. Thomas Lisowsky. (2005). Nucleic
acid-free matrix: Regeneration of DNA binding columns. Biotechniques. 39(2): 270-
271.
Marko MA, R. Chipperfield, H.C. Birnboim. (1982). A procedure for the large-scale isolation
of highly purified plasmid DNA using alkaline extraction and binding to glass
powder.
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M Analyt Biochem 121(2) : 382–387.

TO
AND Osamu Yamada, Toshiya Matsumoto, Masahiro Nakashima, Shinobu Hagari, Toshio
b0 ex Kamahora, Hiroshi Ueyama, Yuichiro Kishi, Hidetoshi Uemura, Takashi Kurimura.
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(1990). A new method for extracting DNA or RNA for polymerase chain reaction.J Virolog Methods .
27(2): 203–209.
Valter de Oliveira LF, da Luz Wallau G., Silva Loreto EL (2009). Isolation of high quality
ADN: a protocol combining “rennet” and glass milk. Electronic J Biotech 12(2)
http://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/v12n2-4/709
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Purification of chromosomal DNA and plasmid

DNA.
Aim .
Know, perform and compare the purification procedure for chromosomal DNA and
plasmid DNA.
Introduction.
The purification of nucleic acids is an essential requirement to perform various

molecular biology techniques. Successful obtaining of reliable and reproducible data
depends, to a large extent, on the extraction of whole DNA and its purification. Extraction
consists of the isolation and purification of DNA molecules and is based on the
physicochemical characteristics of the molecule.
DNA is made up of two chains of nucleotides linked together forming a double

helix. Nucleotides are made up of a sugar (deoxyribose), a phosphate group and a
nitrogenous base (adenine, guanine, thymine or cytosine). The union of the nucleotides
occurs between the phosphate group and the sugar, through phosphodiester bonds, giving
rise to the skeleton of the molecule. The bases of opposite chains are joined by hydrogen
bonds and keep the helical structure stable. The phosphate groups are negatively charged
and polar, which gives the DNA a net negative charge and makes it highly polar,
characteristics that are used for its extraction. Phosphate groups have a strong tendency to
repel each other, due to their negative charge, which allows DNA to dissolve in aqueous
solutions and form a hydrating layer around the molecule. But, in the presence of ethanol,
the hydrating layer breaks down and the phosphate groups are exposed. Under these
conditions, binding is favored with cations such as Na+, which reduce the repulsive forces
between the nucleotide chains and allow the DNA to precipitate. On the other hand, DNA's
net negative charge allows it to bind to positively charged inorganic molecules and
matrices.
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The classic method for purifying nucleic acids is based on the use of toxic organic
solvents, with phenol/chloroform extraction being the best known. However, alternative
methods have recently been developed that use a high salt concentration instead of
organic solvents. The first step consists of the lysis of the cells whose DNA is to be purified.
This step is carried out using an anionic detergent that solubilizes the cellular components.
Said cell breakage is carried out in the presence of “preservatives”, which guarantee the
integrity of the DNA; that is, they prevent or limit the action of the DNases present in the
cells or contaminants in the medium. The RNA present is removed by RNase treatment.
Subsequently, proteins and other cellular contaminants are eliminated by means of

salt precipitation (which replaces the classic treatment with toxic organic solvents). Finally,
genomic DNA is isolated by precipitation in the presence of alcohol and dissolved in water
or a buffered solution that may contain DNA “preservatives.”
There are various procedures for DNA purification, although they all include the
following three steps: (i) Growth of bacteria in a medium selective for those that carry the
plasmid. (ii) Lysis of the bacteria for the release of the plasmid. (iii) Purification of plasmid
DNA.
The main problem to be solved is the separation of plasmid DNA and chromosomal
DNA.
Requirements :
Orbital incubator. Sterile test tubes. GTE isotonic solution
Automatic micropipettes. 1.5 mL Eppendorf tubes. lysis solution
Horizontal electrophoresis Micropipette tips Neutralization solution

chamber. 10, 200 and 1 000 uL with filter
Power supply. Gloves. absolute ethanol
Thermostatted bathroom. Crushed ice. Ethanol 70%
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Vortex shaker. TE pH 8, with Ribonuclease

Rack for 1.5 or 2 mL tubes
TO
UV transilluminator. Paper towels Chloroform
Permanent markers. Paper towels Isopropanol
Marker of weight
Used Tip Containers
1 Kb molecular
APR
Proteinase K
RNase A
Protocol.
Purification of bacterial genomic DNA
a. Add 0.5 mL of bacterial suspension (i.e., stationary phase culture grown overnight)
to a 1.5 mL Eppendorf tube and immerse it in crushed ice.
b. Centrifuge at 13,000–16,000 g for 5 seconds to pellet the cells. Remove as much of
the supernatant as possible: first by decantation; then letting the tube drain on clean
absorbent paper, or removing remains with a micropipette.
c. Add 300 µL of the lysis solution and gently pipette up and down until the cells are
resuspended again.
d. Incubate the sample at 80ºC for 5 minutes to lyse the cells.
e. Cool the sample to room temperature.
R RNA digestion.
c
AND F. Add 1.5
uL of the RNase A solution to the cell lysate.
C cu
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g. Mix the sample well, inverting the tube 25 times.

h. Incubate at 37ºC for 15–60 minutes.
Protein precipitation
i. Cool the sample to room temperature. If it is higher than 21ºC, cool on ice.
j. Add 100
uL of the protein precipitation solution to the cell lysate previously
treated with RNase.
k. Shake vigorously (eg. e.g., with a vortex mixer) at high speed for 20 seconds, to
evenly mix the protein precipitation solution with the cell lysate.
l. Centrifuge at 13,000–16,000 g for 3 minutes.
Note : The precipitated proteins should form a compact pellet.
DNA precipitation
m. Decant the supernatant (containing the DNA) into a 1.5 mL Eppendorf tube
containing 300
uL of pure (100%) isopropanol (also called 2-propanol). The
precipitate (containing the proteins) can be thrown away.
n. Mix the sample by gently inverting 50 times.
o. Centrifuge at 13,000–16,000 g for 1 minute. The DNA should be visible as a small
white precipitate.
p. Remove the supernatant by decanting and drain the tube on absorbent paper.
q. Add 300 uL of 70% ethanol. Invert the tube several times to clear the DNA
precipitate.
r. Centrifuge at 13,000–16,000 g for 1 minute.
s. Carefully remove the ethanol by decanting.
t. Drain the tube on clean absorbent paper and air dry the sample for 15 minutes.
u. Rehydrate the DNA at room temperature overnight.
v. Store DNA at 2–8 ºC (~4 ºC) until use.
28
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Plasmid DNA purification
Sample collection
1. Take 1.5 mL (2 x 750

uL) of a stationary culture of a bacteria carrying a
plasmid with an antibiotic resistance gene.
2. They are dispensed into an Eppendorf tube and centrifuged at room temperature for
3 minutes at 12,000 g.
3. The supernatant is removed (2 x 750

uL) very carefully and THROW IT OUT. A
centrifuge pulse is given, the supernatant is carefully removed and discarded. We
are left with the precipitate of the bacteria.
Process by alkaline lysis
4. Resuspend (with vigorous shaking) the bacteria pellet in 100

u
L of an isotonic solution (GTE: Glucose, Tris and EDTA) at 4°C. Leave it at room
temperature for 5 minutes.
5. 200 are added to the same tube

uL of the lysis solution (NaOH, SDS) that is at
room temperature and freshly prepared. Shake gently by hand by inverting the tube
(about 10 times). Incubate for 5 minutes at 4°C.
Neutralization
6. They are added to the same tube, 150

uL of the neutralization solution (3M potassium
acetate, pH 4.8). It is shaken by hand by inverting the tube (about 10 times).
Incubate for 5 minutes at 4°C.
Plasmid isolation
7. The macromolecular aggregates are precipitated by centrifugation (15 minutes at

12,000 g and 4°C), forming a white, milky-looking sediment. The tube is carefully
transferred to the work table.
8. Removed (2 x 200
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uL) carefully remove the supernatant with the plasmid DNA and
dispense it into a clean tube (previously marked).
9. 1 mL of absolute ethanol (4°C) is added to the tube in which we have placed the
solution with the plasmid DNA. It is mixed by hand by inverting the tube (about 10 times).
Incubate for 15 minutes at 4°C.
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10. The plasmids are precipitated by centrifugation (15 minutes at 12,000 g and 4°C),
removed (2 x 750
uL) carefully remove the supernatant and discard.
11. 900 μl of 70% ethanol (4°C) are added to the same tube. Mix gently by hand by
inverting the tube (about 10 times).
12. The plasmids are pelleted again by centrifugation (10 minutes at 12,000 g and 4°C),
the supernatant is carefully removed and discarded.
13. A centrifuge pulse is given, the supernatant is carefully removed and discarded. We
are left with the precipitate of the plasmids.
14. The plasmid precipitate is dissolved (aspirating and releasing the liquid several times
with the help of the micropipette) in 50
uL of TE buffer (pH 8.0), with
ribonuclease A.
15. Incubate at room temperature for 5 minutes. Keep the sample at 4°C
Results to document
1. Observe and document the types of total DNA extraction of each of the methods.
2. Observe and document comparisons of chromosomal DNA and plasmid DNA
3. Compare the DNA extraction results with other methodologies carried out.
Questionnaire
1. What methods exist to extract and purify DNA?
2. Why is it necessary to carry out the independent extraction of chromosomal DNA
and plasmid DNA?
3. What elements and what cellular substances can degrade genetic material?
4. Why is neutralization solution necessary in DNA extraction and purification
processes?
•
30
GTE isotonic solution:

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50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0.
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• Lysis solution:
0.2 N NaOH, 1% SDS, prepare fresh before each plasmid DNA extraction.
• Neutralization solution:
3 M potassium acetate, pH 4.8.
• TE pH 8.0 with Ribonuclease A:
10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, Ribonuclease A 0.5 μg/μL.
• TAE:
40 mM Tris-Acetate, 1 mM EDTA.
Bibliography.
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds)
(2005): “Current Protocols in Molecular Biology”. Vols 1 to 4. New York: Greene &
John Wiley (New York).
Advise J. c. 2004. Molecular markers, natural history and evolution. Sinauer Associates,
Inc. Publishers. Massachusetts, USA
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening
recombinant plasmid DNA. Nucleic Acids Research 7: 1513-1523.
Dundass N., N. K. Leos, M. Mitui, P. Revell and B. b. Rogers. 2008. Comparison of

automated nucleic acid extraction methods with manual extraction. Journal of
Molecular Diagnostics 10: 311-316.
Stormer M., K. Kleesiek and J. Dreier. 2007. High-Volume Extraction of Nucleic Acids by
Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood
Components. Clinical Chemistry 53: 104–110.
Tokuda Gaku, Nathan Lo, Aya Takase, Akinori Yamada, Yoshinobu Hayashi and Hirofumi
Watanabe. Purification and partial genome characterization of the bacterial endosymbiont
Blattabacterium cuenoti from the fat bodies of cockroaches BMC Research Notes 2008,
1 :118p.
31
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DNA quantification in agarose gels.
Aim.
Estimate DNA concentration by comparing the density of bands on an agarose gel
Introduction .
There are a wide variety of DNA extraction and purification methods, the choice of
method will depend on the origin of the sample, the degree of purity and quality required for
subsequent use.
After DNA extraction, quantification and confirmation of the integrity of the

molecules obtained are necessary. If it is not possible to quantify the DNA by
spectrophotometric methods, the concentration estimation can be carried out by visualizing
it in agarose gels.
Agarose gels are prepared by melting agarose in the presence of the appropriate
buffer until a clear solution is achieved. The molten solution is placed in a mold where it
gels. The gel forms a matrix whose density is determined by the concentration of agarose.
When an electric field is applied through the gel, the DNA migrates towards the anode
through the phosphate groups that confer a net negative charge on these molecules. The
migration speed of DNA molecules is a function of their size, the conformation of the
molecule, the concentration of the agarose and the voltage applied to the electrophoresis
chamber.
The DNA molecules once migrated in the agarose matrix can be visualized by
staining with different dyes, the most commonly used being ethidium bromide. This dye is intercalated
into the DNA and when UV light shines on the agarose gel, a fluorescence is emitted which is
proportional to the amount of DNA. The estimation of the DNA concentration will be obtained by
comparing the fluorescence emitted q by the problem sample with the fluorescence emitted by
molecular weight standards.
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Fig. 1 Horizontal electrophoresis chamber
Requirements:
Horizontal electrophoresis 1 set of P1000, P200 and P10

Agarose solution 1% w/v
chamber micropipettes
Power supply Tips of different Marker of weight

capacities (10, 200 and 1000 µL) molecular (100bp)
Transilluminator Ethidium bromide solution

(10 mg/mL)
Load buffer
1X TAE Buffer
Deionized water
DNA samples
Protocol .
1. Weigh 0.5 g of agarose and mix it in a 250 mL flask with 50 mL of 1X TAE

buffer. Melt the agarose solution in a microwave oven (in 30 second intervals) until the
solution appears completely clear.
2. Prepare the electrophoresis chamber with the appropriate comb depending on the
number of samples.
3. Pour the agarose solution into the electrophoresis chamber and allow the gel to
polymerize.
4. Remove the comb and add TAE 1X buffer to the chamber reservoir.
33
electrophoresis
Page
5. Place 2
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uL of the DNA test samples and the known molecular weight marker
previously mixed with 2
uL of loading buffer inside the wells
Fig. 2 Scheme for the preparation of agarose gel
6. Connect the electrophoresis chamber to the power source and start running at 90V
for a period of 45 minutes to 1 hour.
7. Prepare a solution of ethidium bromide at the concentration (10 mg/mL) in a
volume of 100 mL.
Caution : Ethidium Bromide is a powerful and toxic mutagen. Avoid inhalation of
dust. Wear gloves when working with BrEt solutions.
8. Immerse the gel in the ethidium bromide for 10 seconds.
9. Rinse the gel in water to remove excess ethidium bromide.
10. Place the gel on the transilluminator and turn on the UV light lamp, placing the
acrylic screen to protect against UV light.
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11. Obtain an image of the gel.

12. Compare the density of the DNA band with the molecular weight marker bands.
13. And estimate the concentration.
1. Observe and document the physical characteristics of the DNA sample to be
quantified.
2. Observe and document the advantage of using load buffering in this protocol.
3. Document any conditions during electrophoresis that may make it difficult to
visualize the DNA sample.
Questionnaire .
1. Mentions other DNA quantification methods.

2. What other dyes are there to visualize DNA?
3. What advantages does ethidium bromide have over other dyes?

• 1X APR
Tris 2.42g
EDTA 0.373g
Ac. Acetic 0.572 mL
Make up to 500 mL with deionized water and adjust pH =8

• Load buffer
Bromophenol blue 0.25%
• ilene cyanol FF 0.25%

35
Ficoll (Type 400) 15%

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Note :
a. Ethidium Bromide has mutagenic properties at very low concentrations (up to 0.01
μg/mL), so extreme precautions must be taken when handling it and proper
management of the waste generated.
b. UV light and/or UV radiation is dangerous and can cause damage to the retina of
the eyes. Unprotected UV light should never be directly viewed. UV light is also
mutagenic and carcinogenic. To minimize the risk of exposure, the UV light source
must be adequately shielded. Wear appropriate protective gloves when holding
materials under a UV light source.
Bibliography
Sambrook, J. and Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.).
NY. USA: Cold Spring Harbor Laboratories Press.
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DNA quantification by spectrophotometry
Aim .
Know the quality and quantity of DNA obtained through spectrophotometry.
Introduction .
The spectrophotometer is based on the transmission of light through a solution to
determine the concentration of a solute present in it. Each molecule absorbs radiant energy
at a specific wavelength, from which it is possible to extrapolate the concentration of a solute
in a solution. Proteins and nucleic acids absorb light in the ultraviolet range, at wavelengths
between 210 and 300 nm, the maximum absorbance of DNA and RNA solutions
corresponds to 260. Since DNA and RNA solutions partially absorb light at 280 nm and
those containing proteins do the same at 260 nm, the quotient of the values obtained at 260
nm and at 280 nm (A 260 / A 280 ) provides an estimate of the degree of purity of nucleic
acids. The respective A 260 /A 280 ratios of pure DNA and RNA are approximately 1.8 and
2.0. With a light path of 10 mm and a wavelength of 260 nm, an absorbance A = 1
corresponds approximately to 50 µg/ml double-stranded DNA, 37 µg/ml single-stranded
DNA, 40 µg/ml RNA or 30 µg /ml of oligonucleotides.
To quantify the amount of material obtained after a nucleic acid extraction or the
chemical synthesis of an oligonucleotide, a reading must be taken at wavelengths of 260 nm
and 280 nm. Reading at 260 nm will allow the concentration of nucleic acids in the sample to
be calculated.
It is taken as a reference that an OD (Optical Density), measured at 260 nm,

corresponds approximately to:
150 ug/ml double-stranded DNA,
240
ug/ml single-stranded RNA or DNA
37
320
ug/ml when it comes to oligonucleotides.
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The ratio of the reading between 260 and 280 (OD 260/OD280) provides an
estimate of the purity of the nucleic acid. This is how pure DNA preparations have OD
260/OD280 of 1.8. When it comes to RNA the OD 260/OD280 value is very close to 2.0. If
there is contamination with proteins or phenol or other solutions used during extraction, the
ratio value is less than 1.8 and it is not possible to accurately quantify the DNA.
Requirements.
Microcentrifuge quartz cuvette Tris HCl, EDTA
spectrophotometer Sterile tips Sterile deionized water
Vortex Rack TE Solution
Micropipette Tubes
Gloves, cleaning materials
Protocol .
1. Take the DNA under study and make a 1/10 dilution in TE buffer. Prepare 1ml of
this dilution.
2. Place the DNA solution in the quartz cuvette. This cuvette must have been
previously washed with 1 ml of diluted extran, distilled water and purged with TE
solution. If necessary, add absolute ethanol to achieve complete drying.
3. In another cuvette, place the TE as a blank solution if the DNA was rehydrated with
this buffer or distilled water if this was the hydration solution.
4. Carry out the measurement at 260 nm and 280 nm .

38
5. To determine purity, the DNA/protein ratio is used. The abundance of residual

Page
proteins in the extract is determined by measuring at 280 nm.
6. Carry out the respective calculations, using the following equation to estimate the
biomass of the sample:
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µg/ml DNA = (A 260 X Dilution factor X 50 µg DNA/ml)
7. Estimation of total DNA:
µg DNA/ml or g = (A 260 X 20 X 50µg DNA/ml) / (sample vol. (ml or g))
Results to document
1. Observe and document total DNA estimation data for each sample
2. Observe and document DNA purity comparisons.
3. Document the results by plotting the absorbances obtained from the DNA
sample(s);
4. Compare the DNA quantification results with other methodologies carried out.
Questionnaire.
1. Why do you think it is necessary to estimate the concentration of isolated DNA?
2. Why should sample dilution be performed before measuring OD? in the

spectrophotometer?
3. Why is only the DO taken into account? at 260 nm in the estimation of DNA
concentration? What information does the DO provide us? at 280nm?
4. Compare your results (DNA concentration and purity ratio OD260:DO280) with those of
the rest of the group. aAbs260 Abs280 Abs260/Abs280
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Bibliography.
Catalogs and technical sheets 2012 from commercial companies: Invitrogene, Promega,
Biorad, Biologend, Bioline, Roche.
Lizcano Losada F. 2005. Molecular foundations in medicine. Modern Manual. Savannah

College.
Solano F. G., Márquez C MP, Shuler I. 2009. Optimization of DNA extraction from
Passiflora ligularis for analysis using molecular markers. Universitas Scientiarum
14 (1) 16-22.
Vargas R., Rangel LF Cañola E. A., Rodriguez J. AND. L., Kilger JC, Bastidas G. A.E.
2006. Detection and quantification of circulating DNA levels in human serum using
UV-Visible spectrophotometry. Rev. Colombian Physics 38(2) 946-949.
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Extraction by bacterial RNA kit.
Aim .
1. Extract RNA from prokaryotic cells using a commercial procedure as well as

acquire basic knowledge for the management and care of the extracted RNA
Introduction .
Most protocols for RNA extraction are based on the method described by
Chomezynski and Sacchi, which is based on the isolation of this nucleic acid using phenol-
chloroform and guanidine isothiocyanate. Each of these components plays a specific role:
guanidine thiosanate acid is extremely strong and has a high denaturing power; The
phenol, being acidified, causes the DNA to accumulate at the interface between the
aqueous phase and that of the phenol, leaving the RNA in the aqueous phase. Due to its
chemical structure, RNA is a very fragile molecule that can be broken by the action of the
2'-OH groups (highly reactive) adjacent to the ribose-phosphate backbone.
The greatest difficulty in obtaining RNA preparations is that the majority of RNases,
which degrade our molecules of interest, are very stable and active enzymes, not requiring
cofactors for their operation. A small amount of them that remains as a contaminant in our
preparation can constitute a real problem. Therefore, it is important to treat samples with
RNase-free DNases.
There are commercial packages for stabilization of RNA samples from companies
such as QIAGEN, Ambion and RNA-works, which generally work with a mixture of
ribonuclease-specific antibodies, ribonuclease inhibitors and/or contain compounds that
neutralize 2'-OH groups. (as is the case with RNA-works). We will use the commercial ZR
Fungal/Bacterial RNA Micropipet Kit (Zymo Research) to carry out this practice.
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Requirements :
Microcentrifuge 1.5mL eppendorf tubes ZR Fungal/Bacterial RNA

Micropipet (Zymo Research) (3)
Vortex Micropipettes of several MgSO4• 7H 2 O 10mM

volumes sterile
Ethanol 95-100%
Laminar Flow Hood Blue, yellow and white tips
Bacteriological loop
Lighter
Protocol.
2. One day prior to the practice, a colony of E cells is inoculated. coli in 5 mL of LB

broth using the respective antibiotic. Incubate at 37ºC overnight with shaking.
3. The next day, resuspend the bacterial cells that grew the previous day in the
Eppendorf tubes in sterile 10 mM MgSO4•7H 2 O. Vortex and centrifuge at
maximum speed. Decant.
4. Resuspend cells at 800
uL in RNA Lysis Buffer and transfer the
mixture to a ZR Bashing Bead Lysis tube.
5. Assemble the above device in a Bead beater tube and centrifuge for 1 minute at
12,000 xg.
6. Transfer 400
uL of the supernatant to a Zymo-Spin IIIC column fitted to a
collection tube and centrifuge at 8,000 xg for 30 seconds (reserve the collected
flow in the collection tube).
7. Add 0.8 volumes of 95-100% ethanol and vortex well.
8. Transfer the mixture to a Zymo-spin IC column prepositioned inside a collection tube.
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9. Add 400 uL of RNA Prep Buffer to the column. Centrifuge at 12,000 x g for 1
minute. Discard the collected flow and place the Zymo-spin IC column into the
same collection tube.
10. Add 800 uL of RNA Wash Buffer to the column. Centrifuge at 12,000 x g for 30
seconds. Discard the collected flow and place the Zymo-spin IC column into the
same collection tube. Repeat this step with 400m
uL of RNA Wash Buffer.
11. Centrifuge the Zymo-spin IC column at 12,000 xg for 2 minutes. And make sure to
remove all the wash buffer.
12. Remove the Zymo-spin IC column from the collection tube and place it into a 1.5
mL DNase/RNAase-free Eppendorf tube (sterile).
13. Add ≥6
uL of DNAase/RNAase-Free water directly to the column matrix and
let stand for 1 minute.
14. Centrifuge at 10,000 x g for 30 seconds to elute the RNA from the column.
15. The RNA can be used immediately or can be stored at -70⁰C
16. Label the eppendorf tube
Vortex Fungal/Bacterial Sample With ZR

with the equipment number.
BashingBeadtu Lysis Tube
Fitter Lysate
With Zymo-Spintu IV
Bind, Wash. Elute DNA

With Zymo-Spinmu IIC
PCR Ready Ultra-pure DNA
Diagram: ZR Fungal/Bacterial
Page
RNA MicroPrep TM.
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1. Observe and document the phenotypic characteristics of both plate and culture
cultures.
in tube.
2. Observe and describe the step of the protocol that must be taken care of to prevent
the RNA
becomes contaminated and degraded.
3. Document the performance and quality of the sample obtained by using
of a commercial kit and compare it with the performance obtained using a non-
commercial protocol.
Questionnaire
1. Mention three advantages and disadvantages of using a commercial kit or

traditional protocol.
to extract RNA.
2. Research some steps to consider so that during RNA electrophoresis
prevent degradation of the sample.
3. Investigate in which molecular biology tests purified RNA can be used.
Bibliography
Chomczynski P. and Sacchi. (1987). Anal Biochem. 162:156.
Falcon LI and Valera A. (2007). Molecular Tools (fifth part). Nucleic acid extraction:
Chapter 16. Molecular Ecology. Luis E. Eguiarte, Valeria Souza and Xitlali Aguirre
(Compilers).
ZR Fungal/Bacterial RNA MicroPrep TM. Instruction Manual. ZymoResearch.
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Page
Conclusion of the manual.
The purification and analysis methodologies of nucleic acids are basic and very important
in any Molecular Biology procedure, so this manual will be of great help to the student who
is beginning to develop skills and abilities in the subject.
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General Bibliography.
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds)
(2005): “Current Protocols in Molecular Biology”. Vols 1 to 4. New York: Greene &
John Wiley (New York).
Advise J. c. 2004. Molecular markers, natural history and evolution. Sinauer Associates,
Inc. Publishers. Massachusetts, USA
Balbas P. (2010). From Molecular Biology to Biotechnology (2nd. edic). Mexico: Trillas.
Bert Vogelstein and David Gillespiet. (1979). Preparative and analytical purification of DNA
from agarose. Proc. Natl. Academic Sci. USA 76(2): 615-619.
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening
recombinant plasmid DNA. Nucleic Acids Research 7: 1513-1523.
Catalogs and technical sheets 2012 from commercial companies: Invitrogene, Promega,
Biorad, Biologend, Bioline, Roche.
Chomczynski P. and Sacchi. (1987). Anal Biochem. 162:156.
Debra A. Dederich, Geoffrey Okwuonu, Toni Garner, Amanda Denn, Angelica Sutton,
Michael Escotto, Ashley Martindale, Iliver Delgado, Donna M Muzny, Richard A
Gibbs and Michael L. Metzker. (2002). Glass bead purification of plasmid template
DNA for high throughput sequencing of mammalian genomes. Nucl Acds research
30(7): 1-5.
Dundass N., N. K. Leos, M. Mitui, P. Revell and B. b. Rogers. 2008. Comparison of

automated nucleic acid extraction methods with manual extraction. Journal of
Molecular Diagnostics 10: 311-316.
Etienne Joly. (1996). Purification of DNA Fragments from Agarose Gels Using Glass
Beads. Basic DNA and RNA Protocols. Methods Mol Biol. 58: 237-240.
Falcon LI and Valera A. (2007). Molecular Tools (fifth part). Nucleic acid extraction:
Chapter 16. Molecular Ecology. Luis E. Eguiarte, Valeria Souza and Xitlali Aguirre
(Compilers).
Page
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Green MR and Sambrook J. (2012). Molecular cloning: A Laboratory Manual (4th edition).
Woodbury, New York: Cold Spring Harbor Laboratory Press.
Hamid Kheyrodin1 and Khosro Ghazvinian. (2012). DNA purification and isolation of
genomic DNA from bacterial species by plasmid purification system. African J
Agricultural Research 7(3): 433-442.
Karl-Heinz Esser1, Dr. Wolfram H. Marx2 and Prof. Dr. Thomas Lisowsky. (2005). Nucleic
acid-free matrix: Regeneration of DNA binding columns. Biotechniques. 39(2): 270-
271.
Lizcano Losada F. 2005. Molecular foundations in medicine. Modern Manual. Savannah

College.
Madigan MT, Martinko JM, Bender KS, Buckley D. H.,, Stahl D. TO. and Brock T. (2015).
Marko MA, R. Chipperfield, H.C. Birnboim. (1982). A procedure for the large-scale isolation
of highly purified plasmid DNA using alkaline extraction and binding to glass
powder. Analyt Biochem 121(2) : 382–387.
Osamu Yamada, Toshiya Matsumoto, Masahiro Nakashima, Shinobu Hagari, Toshio

Kamahora, Hiroshi Ueyama, Yuichiro Kishi, Hidetoshi Uemura, Takashi Kurimura.
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Laboratory Practice Manual for Introduction to Molecular Biology I

Laboratory Practice Manual

Bachelor's degree in Biotechnology

Ricardo Carreño López

Fabiola Avelino Flores

María del Rocío Bustillos Cristales

Edith Chavez Bravo

Rocío Pérez and Terrón

Lorena Milflores Flores

General rules for the use of the Biology laboratory Molecular.................................................7

General rules for the use of the Biology laboratory

ventricular fibrillation with possible temporary cardiac and respiratory paralysis. In

In general terms, a current, either direct or alternating, of 100 mA is dangerously lethal.

Genomic DNA Extraction: Traditional Method and Kit

• The desired results (yields, purity, purification time)

• The subsequent application (amplification, cloning, expression, reverse transcription).

• Mechanical disruption, such as hypotonic disruption, or freeze-thaw.

• Chemical treatment, such as lysis with detergents or chaotropic agents.

• Enzymatic digestion with proteases, lysozyme or mutanolysin.

• Extraction/precipitation using solvents to remove contaminants, such as combining phenol and

• Centrifugation, which is an important purification method frequently used in combination with

Equipment Material Reagents

Extraction hood 15 mL falcon tubes Medium LB

Vortex Sterile double-distilled water or

Microcentrifuge Micropipette from 10 to 100 3M Sodium Acetate with pH

Extraction hood Strain of E. coli K-12 absolute ethanol

Freezer at -20°C Blood 1 mL Chloroform

Water bath Zymo Research Quick-gDNA TE Buffer pH 8.0

Extraction of genomic DNA from an Escherichia coli strain.

1. Propagate a culture of E. coli in LB medium (5 mL) overnight (18-24 hours)

2. Place 1 mL of the culture in a 1.5 mL polypropylene tube, centrifuge the tube at

3. Add 250 µL of water sterile deionized to cell pack and shake

5. Add 250 µL of chloroform and shake vigorously using a vortex for

6. Centrifuge at 9,000 rpm for 5 minutes.

8. Add 225 µL of chloroform and shake vigorously for 30 s using a vortex.

9. Centrifuge at 9,000 rpm for 5 minutes.

13. Remove the supernatant and let it dry at 37ºC.

Blood Sample + Genomic Lysis

Ultrapure DNA for: PCR

1. What DNA extraction processes were used in each case?

5. What would be the applications of these techniques in Biotechnology?

Preparation of solutions and reagents.

1. Phenol balanced with 1 M Tris pH 8.0

ZymoResearch. (2014). Quick-gDNA MiniPrep Kit Instruction Manual

Extraction of plasmid DNA by traditional method

Isolate plasmid DNA from prokaryotic cells using a traditional method.

Most plasmids are characterized by:

• Have an autonomous replication origin.

Equipment Material Reagents

Shaking incubator 15 mL falcon tubes LB Broth

Microcentrifuge 1.5 mL eppendorf tubes Birnie I Solution

Vortex Birnie II Solution

New, sterile blue, yellow and

Vacuum concentrator Ice Cold absolute isopropanol

cold absolute ethanol

Sterile double-distilled water or

1. Centrifuge 1.5 mL of a 24-h bacterial culture at 8000 rpm for 2 minutes.

5. Centrifuge at 12000 rpm for 10 minutes. Add 600

6. Centrifuge at 14000 rpm for 10 minutes.

7. Remove the supernatant and wash the package with 1000

9. Resuspend the DNA in 50 uL of sterile double-distilled water and store at -20°C.

1. What is the purpose of neutralization in the “alkaline lysis” method?

4. What uses can be given to plasmids in Biotechnology?