BIOL 336 bacterial genetics
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Lab 1: Basic Techniques
LAB 1: BASIC TECHNIQUES
INTRODUCTION:
The studies of bacterial genetics in the lab often require growing, isolating, and transferring
bacterial cultures. Good aseptic techniques and streak for isolation are skills that you will need
to accomplish these tasks. In addition, many experiments in bacterial genetics also involve the
use common molecular biology techniques such as gel electrophoresis and restriction digestion.
In this lab period, you will have the opportunity of reviewing and practicing those skills.
OBJECTIVES:
1) Extract plasmid from bacteria using simple (not commercial kit) protocols (boiling miniprep
and minute miniprep) and examine the extracted DNA.
2) Determine and calculate the amount of components require to prepare an agarose gel to
analyze specific size range of DNA and set up and perform agarose gel electrophoresis
3) Determine and calculate the amount of components to be used to perform at restriction
endonuclease digestion of a specific amount of DNA.
4) Perform an assigned restriction endonuclease digestion of a specific amount of DNA
5) Demonstrate the usage of aseptic techniques for the inoculation of broth and plates by
successfully inoculating different types of media and producing/maintaining pure cultures
6) Produce culture plates with distinctly isolate colonies from a given bacterial culture using the
streak for isolation technique
RESOURCES:
Lab manual from BIOL 210 Microbiology 1, lab notebook
ACTIVITIES:
1) Brief Presentation about isolating plasmids from bacteria. Outline of the procedure and
application of the boiling plasmid miniprep.
2) Exercise 1: Boiling Rapid Plasmid Miniprep
3) Brief presentation about making agarose gels for electrophoresis
4) Exercise 2: Agarose Gel Electrophoresis
5) Brief presentation about setting up a digestion of DNA using restriction endonucleases
6) Exercise 3: Restriction digests of DNA
7) Exercise 4: Practice of aseptic technique & Streak for isolation
EXERCISE 1: FAST PLASMID MINIPREPS (no kit)
OBJECTIVES:
Describe the principles used to design methods to extract plasmid from bacteria
Explain in which circumstances a particular plasmid extraction technique may be used
I. PLASMID MINIPREP BOILING TECHNIQUE (Reference: Ausubel et. al. 1997
Short Protocols in Molecular Biology Third Edition Wiley & Sons, Inc. New York)
A very quick procedure used for preparing small amounts of plasmid DNA from a large number
of samples (up to 24 samples can be processed very fast) It is very useful for initial evaluation
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Lab 1: Basic Techniques
of clones. Other methods, like the alkaline lysis plasmid miniprep that we will be doing on a
future lab produce a higher quality material
I.1.b. MATERIALS:
O/N culture of bacteria (5 ml). I will be aspecific E. coli bacterial strain (SK(-), sk700, sk1640,
pWR501, DH5α )
Microcentrifuge and 1.5 ml sterile microcentrifuge tubes
Boiling water bath (hot plate, beaker, heat resistant floating rack)
Ice and ice buckets
System for drying under vacuum
Access to a - 20C freezer
Microfuge tube racks
STET solution (recipe below)
Hen egg white lysozyme
Isopropanol Ice cold
TE buffer
10 mg\ml of Rnase solution (DNAse free) OPTIONAL
I.1.c. PROCEDURE: (students star at step 2)
1) Inoculate 5m of LB medium with a single colony of the bacteria that may contain the
desired plasmid. Let it grow to saturation (usually 16 hours or o/n). Note: this step will
be done for you by the instructor or lab technician.
2) Spin down 1.5 ml of the culture in a microcentrifuge for 20 seconds. Discard
supernantant in proper container. Resuspend the bacterial pellet very well by vortexing
in 300 µl of STET solution containing 200 µg of lysozyme. Place tube for about 5
minutes on ice (can stay as long as 10 min) NOTICE: alternative to vortexing is to slide the
microtube back and forth on the surface of a solid microfuge tube rack
3) Place the tube in a boiling water bath (100 C) for 2 minutes, centrifuge for 20
minutes. Remove the supernatant into a new tube
4) Mix the supernatant with 200 µl cold isopropanol and place 30 minutes at - 20 C
5) Centrifuge 5 minutes, discard supernatant, and dry pellet under vacuum
6) Resuspend in 50 µl of TE buffer and store in the fridge until needed. You will need
to use a 3 l aliquot of this material for observation of uncut plasmid DNA and
about 5 µl for each restriction digest.
7) If required, you may remove contaminant RNA by adding 1 µl of 10 mg\ml of Rnase
solution (DNAse free). The plasmid should be store at 4 C for short-term usage and 20C or - 70C for long term usage
8) Evaluate your plasmid by agarose gel electrophoresis
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Lab 1: Basic Techniques
I.1.c RESULTS:
Compare your plasmid with the control plasmid and the other groups samples. Determine the
following:
a) How many bands do you see in the gel? Explain the origin of these bands
b) Does your plasmid appear to have an insert? Explain
c) Base on the apparent amount of plasmid DNA you recovered with this procedure estimate
how many different restriction digestions could you do with your prep?
II. MINUTE PLASMID MINIPREP FRO FAS EVALUATION OF CLONES
This protocol has been passed on among graduate students, technicians and postdoctoral
fellows working in labs all over the world. Unfortunately, the name of the individual that first
came up with this protocol has been lost in the exchange. The technique is very simple, cheap
and allows a quick evaluation of many clones.
OBJECTIVES:
Describe the chemical principles that allow extraction of plasmid using the minute
phenol/chloroform technique
Extract plasmid DNA from bacteria using the minute phenol chloroform technique
II.1.b MATERIALS:
the following E. coli bacterial strains will be used:
SK(-), sk700, sk1640, pWR501, DH5α these will be available in plates
TE buffer pH 8
Phenol/chloroform mixture 1:1
Plate with blue and white colony clones to be tested (not older that 48 hours)
Microcentrifuge and tubes for centrifugation
Bunsen burner and inoculating loop
II.1.c PROCEDURE: (students start at step 2)
Note: this procedure is usually performed after a transformation experiment where colonies
carrying a plasmid with an insert are white while colonies that do not carry insert are blue. We
will explain how this works in a future lab. For now you just follow the instructions.
The 1st step for this procedure will be performed by the technician a day prior to the lab.
1) Pick colonies and strike them on a plate containing X-gal/IPTG and grow it overnight
according with the following scheme (see diagram below) and incubate at 37 ◦C overnight
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Lab 1: Basic Techniques
2) Normally you will be only interested in the white clones. However in this experiment one
of you will select a blue clone and the other should select a white clone (so that when running
the gel you can actually see the difference in the size of the plasmids)
3) take a small amount of cells (about a loop-full) of a solid mass of cells
4) resuspend the cells in a microfuge/Eppendorf tube containing 20 μl of T10E1 buffer pH 8
vortex if necessary
5) add 20 μl of phenol/chloroform mixture and vortex
6) centrifuge for 2 minutes at high speed
7) take 5 μl of the supernatant and placed it on a fresh tube
8) place the new tube with the lid open in a 37 ◦C heating block for 5-10 minutes to allow
chloroform residues to evaporate (this is a good time to prepare the agarose gel for observation
of the samples)
9) mix the 5 μl sample with appropriate amount of DNA loading buffer and load it into a 0.8%
agarose gel. Run the gel at about 95 Volts for 45 minutes
II.1.d. RESULTS:
Take a picture of the gel and make your observations. Notice: There will be a picture available on the
web site for you to be able to see how does these kind of miniprep would look like .
Compare your plasmid with the control plasmid and other groups’ samples. Determine the
following:
a) How many bands do you see in the gel? Explain the origin of these bands
b) Does your plasmid appear to have an insert? Explain
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Lab 1: Basic Techniques
EXERCISE 2: AGAROSE GEL ELECTROPHORESIS
In this exercise, you will learn to prepare agarose gels for electrophoresis. During this course
you will use this technique to examine the DNA and RNA material that will be prepared in
future exercises. Time constrictions may result on completion of this exercise the following
week. Your instructor will give you very precise instructions about this in the lab
The instructor will demonstrate the making of the gel today. You will be referring to the
instructions below for every situation in which you require to make an agarose gel
2.b MATERIALS:
Agarose powder
weight boats
scale
microwave oven
50X TAE buffer
10, 100, 1000 ml graduate cylinders
Several size Erlenmeyer flasks
Distilled water
Gel casting trays, combs, and electrophoresis chamber
Ethidium bromide solution (10mg\ml)
termomether
Warning ! Ethidium bromide is an hazardous chemical. Usually the electrophoresis chamber,
casting tray, combs are contaminated due to the use of this chemical. You must wear gloves
when handling all the equipment and the stained gels.
2.c. PROCEDURE:
NOTICE: In this 1st lab you and other groups in your course will run a DNA sample from the minipreps done
in exercise 1. Your instructor will give you the specific details in the lab. The instructions written below apply for
any time that you need to perform agarose gel electrophoresis.
Do the calculations required for STEPS 1 through 3 and write this information down in
your lab notebook before proceeding
1) Determine the volume of buffer necessary for the particular size electrophoresis apparatus
that you will be using
2) Determine the volume of the gel used for the particular casting tray to be used
3) Decide the concentration of the agarose gel that you will be using and how many lanes (one
lane for nucleic acid sample to be examine by electrophoresis including molecular markers) you
will require, so that you will select the appropriate comb. You should also find out how much
volume the wells produced by that comb hold
4) Weight the agarose needed and place it on the bottom of a dry Erlenmeyer beaker. Carefully
add the appropriate volume of 1X TAE buffer
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Lab 1: Basic Techniques
5) heat the agarose \buffer mixture in the microwave oven at low power for a few minutes. You
must observe the process carefully and open and check for melting of the agarose every minute
or so. If the mixture overflows in the oven, discard it and repeat. Be careful! It will be
extremely hot Once the agarose is almost completely melted carefully swirl the flask to mix,
keep heating the mixture until no traces of the agarose are visible.
6) Let the agarose mixture cool down to about 60 C and add about 60- 100 µl of the ethidium
bromide stock solution (wear gloves) mix carefully
7) While waiting have the ends of a casting tray taped. Place the comb in the appropriate slots,
place the gel on a even surface and poor the gel. Leave the gel stand undisturbed until it
solidifies, this will take about 20-30 minutes (depend on concentration).
8) Once the gel is solid. Remove the tape from the ends, remove the comb by gently but firmly
pulling it upwards (do not wiggle it!) While holding the tray down. Place the gel on the
electrophoresis chamber. Make sure the wells are closer to the negative electrode end. Fill the
chamber with 1X TAE electrophoresis buffer until the buffer barely covers the whole surface of
the gel
9) Using a small pipette carefully flush the wells (can use a p20)
10) Load your samples ( the DNA samples should be mixed with DNA loading dye before
loading into gel!) and connect the electrodes to the power pack. Turn the power on and set the
voltage to 100 V. Verify that the system is working by carefully checking for the formation of
the bubbles on the electrodes. Also, you will be notice the loading dye moving from the wells
into the gel. Make sure the samples are going in the right direction!
11) Allow the gel to run until the first blue line (the dye front) has advance at least 2\3 of the
way from the whole gel.
12) Turn off the apparatus. Remove the gel and place it on the transilluminator for observation.
Your instructor will show you how to take pictures for your lab notebook records
13) Every picture should be properly labeled. On the appendix at the end of this lab there are
three diagrams representing three different gels and the proper way to write the information that
should accompany any picture of an electrophoresis experiment.
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Lab 1: Basic Techniques
EXERCISE 3: RESTRICTION ENDONUCLEASES
In this exercise you will digest some DNA samples with specific restriction endonucleases
which you will be using later on as size marker for examination of other DNA materials by
agarose gel electrophoresis
OBJECTIVES:
Calculate amounts of different components needed to digest a specific amount of DNA
Explain what restriction nucleases are and how do they work
Perform a restriction digestion of different DNA molecules
Estimate the size and number of fragments produced in a specific restriction digestion exercise
HOW TO SET UP A RESTRICTION DIGESTION:
There are a few considerations and simple calculations that you must perform in order to set up
a successful restriction digestion:
1) Decide how much DNA you will be digesting. The volume is determined based on the DNA
concentration (g /l) and what you will be using it for
2) Decide the enzyme that you will be digesting the DNA with, and the final volume of the
digestion reaction
3) Find the concentrations of the enzyme and its buffer stock. Calculate how many l of both
will be needed for the reaction.
4) Finally, calculate how much water will be needed to complete the final volume of the
restriction digestion after all the other reactants had been taken into consideration (i.e. DNA,
enzyme, buffer). You also need to find the temperature at which the enzyme works.
IN TODAY’S EXERCISE, you are expected to do the following restriction digestions:
a) 0.4 µg of  DNA digested with one of the following enzymes EcoRI or HindIII (each group
in a bench will be assigned a different enzyme)
b) 0.4 µg of plasmid pBR322 digested with the enzyme EcoRI
c) set up the appropriate control reactions (consult your instructor for this)
3.b. MATERIALS:
Lambda DNA (0.2 µg\µl)
Plasmid pBR 322
1kb ladder (0.1 µg\µl)
Eco RI enzyme and 10X EcoRI buffer
Hind III enzyme and Hind III buffer
microfuge tubes
20, 200, 1000 µl micro-pipettes and tips
Ice bucket
Crushed ice
Sterile distilled water
DNA loading buffer
Incubator at 37 C
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Lab 1: Basic Techniques
3.c .PROCEDURE:
1) Decide how many different reaction tubes you will need set up including a control for each
reaction
2) Your instructor will tell you: a) the final volume of the reactions and b) how much DNA in
µl, and enzyme will be used in each reaction
3) Work out how much 10X buffer (specific for the enzyme), and water has to be added to each
particular reaction
4) Collect the necessary number of sterile microfuge tubes (check with your instructor for size)
and label them according to the experiments that you will be performing
5) Add the appropriate reagents to each reaction tube in the following order:
10X restriction enzyme buffer, DNA, water, Enzyme
5) Mix the contents of the tubes very well, then do a quick spin in the microfuge to collect all
liquid droplets at the bottom of the tube.
6) Incubate the tubes for an hour at the recommended temperature, mix the contents of the tubes
every 15 minutes and repeat step 5 above. Alternatively, the material can be left for over night
digestion.
7) Digested material can be examined by agarose gel electrophoresis or stored in the freezer
until the next lab period. Do not forget to add the appropriate amount of gel loading buffer
before loading the samples into the agarose gel.
3.d. RESULTS:
You must run a sample of your digested DNA using agarose gel electrophoresis to evaluate the
quality of your digestion. If possible, obtain a picture of the agarose gel and attach it to your
lab notebook.
a) Was the digestion successful? If not, discuss the possible sources of error.
b) How many fragments do you see? Do these results agree with the expected results?
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Lab 1: Basic Techniques
EXERCISE # 4: REVIEW OF ASEPTIC TECHNIQUE & STREAK FOR
ISOLATION
4.a BACKGROUND:
Please review the information available in your BIOL 210 Microbiology I Lab Manual about
aseptic technique, streak for isolation, and proper labeling of bacterial cultures.
210-01 (1.a MH)
Sep/20/04 ( N.A.)
E. coli
PROPER LABEL OF A PETRI PLATE
Figure 1.3: Information that should be found in Petri plates
4.b.MATERIALS:
Overnight broth culture of E. coli
One Petri plate per student containing nutrient agar (NA)
Inoculating loop, Bunsen burner
4.c PROCEDURE:
1) Label the bottom of the plate to be inoculated
2) Use the streak for isolation technique learned in BIOL 210 to inoculate a plate containing
nutrient agar with E coli from the broth culture provided
3) Incubate the streaked plate at the indicated temperature in an inverted position to prevent
drops of condensed water falling onto the surface and smearing the colonies
4) Next day examine the plates for isolated colonies. Score your streak for isolation technique
by comparing your plate with those shown in the diagram below
MAKE SURE YOU ARE CONFORTABLE WITH BOTH ASEPTIC TECHNIQUE AND
STREAKING FOR ISOLATION