TEACHER'S MANUAL
EXPERIMENT #10
RESTRICTION MAPPING OF
LAMBDA DNA
Students electrophorese three lambda phage DNA restriction endonuclease digests
and analyze fragment patterns to construct restriction maps of lambda DNA.
Restriction enzymes (RE) have developed into one of the primary tools in
molecular biology. They can be employed to cut DNA molecules precisely into
fragments of a predictable size. A more accurate name for this class of enzymes
is restriction endonuclease, because they break DNA
molecules at internal (endo) positions. Enzymes that degrade DNA by digesting
the molecule from the ends of the DNA strand are called exonuclease (exo =
"outside").
Restriction endonucleases are frequently named using the following convention:
The first italicized letter indicates the genus of the organism from which the
enzyme was isolated. The second and third italicized letters indicate the
species. An additional letter indicates the particular strain used to produce
the enzyme. The Roman numerals denote the sequence in which the restriction endonuclease enzyme from that particular genus, species, and strain of bacteria have been isolated. See Table I.
| Table I |
|
EcoR I
|
E = genus Escherichia
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|
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co = species coli
|
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R = strain RY 13
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I = first RE to be isolated from this species
|
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BamH I
|
B = genus Bacillus
|
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am = species amyloliquefaciens
|
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H = strain H
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I = first RE to be isolated from this species
|
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Hind III
|
H = genus Haemophilus
|
|
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in = species influenzae
|
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d = strain Rd
|
|
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III = third RE to be isolated from this species
|
Some restriction endonucleases cut cleanly through the DNA molecule by
cleaving both of the complimentary strands of the DNA molecule at the same
nucleotide position within the recognition sequence. The recognition sequences
are generally 4 to 6 basepairs long (refer to Table II).
| Table II |
|
Some Restriction Endonucleases and Their Recognition Sequences
|
|
Enzyme Source
|
Abbreviation
Recognition
|
Sequence
|
|
Bacillus amyloliquefaciens H
|
BamH I
|
G/GATCC
CCTAG/G
|
|
Brevibacterium albidum
|
Bal I
|
TGG/CCA
ACC/GGT
|
|
Escherichia coli RY13
|
EcoR I
|
G/AATTC
CTTAA/G
|
|
Haemophilus aeqyptius
|
Hae III
|
GG/CC
CC/GG
|
|
Haemophilus influenzae Rd
|
Hind III
|
A/AGCTT
TTCGA/A
|
|
Haemophilus parainfluenzae
|
Hpa II
|
GTT/AAC
CAA/TTG
|
|
Haemophilus parainfluenze
|
Hha II
|
C/CGG
GGC/C
|
|
Providencia stuartii 164
|
Pst I
|
CTGCA/G
G/ACGTC
|
|
Streptomyces albus G
|
Sal I
|
G/TCGAC
CAGCT/G
|
These 4-8 nucleotide recognition sites are also termed palindromic sequences,
because both strands have the same sequence running in opposite directions.The
RE scans the length of the DNA molecule and stops to cut the molecule only at
its particular recognition site. For example, the endonuclease Hind III will
cut a double strand of DNA in the following way (see page 2):
The Hind III enzyme recognizes the sequence
AAGCTT
TTCGAA
and will cut the DNA as shown below whenever it encounters that sequence.
Some restriction endonucleases (Hae III, Bal I) cut cleanly through the DNA
molecule by cleaving both of the
complimentary strands of the DNA molecule at the same nucleotide position
within the recognition sequence. These enzymes produce a blunt-end cut (Figure
1).
Bal I cuts at the center of the restriction site, leaving blunt ends.
Other RE's (Hind III) cleave the complimentary strands at a different point
within the recognition sequence, which results in a staggered cut. A staggered
cut exposes single stranded regions of the molecule. These single stranded
regions ("sticky ends") are especially useful in making
recombinant-DNA molecules (Figure 2).
Hind III makes staggered cuts in DNA leaving cohesive
"sticky" ends.
DNA restriction fragments produced by the same RE can be spliced together. The
sticky ends produced as a result of the staggered cut by the restriction enzyme
allow complimentary regions in the sticky ends to recognize one another and
pair up. The enzyme ligase, ATP, magnesium ions, and proper pH and temperature
are required to reform DNA bonds that have been broken by a restriction enzyme
that makes a sticky end cut.
Lambda is a bacteriophage which infects E. coli cells. The viral DNA is linear,
double-stranded molecule of 48,502 basepairs (bp) [48.5 Kbp]' with a molecular
weight of about 3 x 10 7 and codes for approximately 50 different phage
proteins. The first 12 bases at both 5' ends are single stranded and can
hybridize to each other to form circular molecules (Figure 3).
Every nucleic acid chain has a direction defined by the orientation of its
sugar-phosphate backbone. The end terminating with the 5' carbon atom is called
the 5' end, while the end terminating with the 3' carbon atom is called the 3'
end. All RNA and DNA chains grow in the 5' --> 3' direction. By convention,
the first base at the left (5'-end) of the linear DNA is nucleotide number 1.
The base sequence of these terminal regions, which are known as cos (cohesive
sticky ends), are complimentary to each other. Thus, by forming base pairs
between the cohesive ends, linear DNA molecules will circularize as shown in
Figure 3.
The circularization of lambda DNA also occurs in an infected
bacterial cell. When lambda phage infects an E. coli cell host, the phage DNA
is injected into the cell and cohesive ends anneal or stick together to form a
circular molecule. Transcription and replication of the phage DNA occurs, and
more phage particles are made within the host bacterium. Later, the bacterial
cell breaks apart (lyses), releasing new infective phages, and the bacterial
cell dies. Less common is the integration of phage DNA into the bacterial
chromosome. Following integration, the bacterial cells behave normally, and
both phage DNA and bacterial chromosomal DNA is replicated together. If the
bacterial cell is subjected to a negative outside environmental insult
(ultraviolet light), the lambda DNA is excised from the bacterial chromosome
and begins its normal viral replicative activities.
The DNA of phage lambda may be divided into three regions (see figure 4). The
left-hand region includes all the genes
(A through J) whose products are necessary to produce phage head and tail
proteins and to package the DNA into the virus. The central region contains
genes involved in the integration of the DNA into the E. coli chromosome. The
remaining portion of the viral genome includes the major control region for
transcription and replication as well as the genes for cell lysis.
Some of the restriction endonucleases that cleave lambda are listed in Table
III below. The positions in the table refer to the 5' base of the recognition
sequence. Site position(s) read left to right (5' --> 3' position).
| Table III |
No. of
Enzyme
|
Sites
|
Position of Sites
|
|
|
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
|
BamH I
|
5
|
5505
|
22346
|
27972
|
34499
|
41732
|
|
|
|
EcoR I
|
5
|
21226
|
26104
|
31747
|
39168
|
44972
|
|
|
|
Hind III
|
7
|
23130
|
25157
|
27479
|
36895
|
37459
|
37584
|
44141
|
For example, a restriction map for phage lambda cut by
Bam H I is presented above (Figure 5).
Beginning at left, each of the numbered restriction sites was plotted along the
length of the phage genome. The number of nucleotides (the fragment size) to
the left of each restriction site were then calculated:
|
(6)
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16,841
|
|
|
7,233
|
|
|
6,770
|
|
|
6,527
|
|
|
5,626
|
|
|
5,505
|
Any restriction enzyme that cuts linear DNA N times will yield N+1 fragments.
Because smaller DNA fragments migrate faster down a gel, the order in which you
would expect to observe fragments ranges from highest to lowest base pair
number.
In this experiment, you will study the effects of EcoR I, Hind III, and EcoR I/
Hind III double digests on lambda DNA, and by analyzing fragment patterns,
construct a restriction map of these restriction sites.
Kit Materials:
See complete materials list for this experiment at the end of this manual.
This kit provides enough material for a class of 30 students (6 groups; 5
students/group).
- DNA Samples:
- lambda DNA / EcoR I digest (2 x 10 ug)
- lambda DNA / Hind III digest (2 x 10 ug)
- lambda DNA Hind III/ EcoR I double digest (2 x 10 ug)
Note: Each 10 ug sample contains enough DNA to run 8 lanes. Therefore, each
sample type contains enough DNA to run a total of 16 gel lanes.
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1
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Bottle, 0.8% pre-cooked agarose (200 mL)
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1
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Bottle, TBE running buffer (10X) (250 mL)
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2
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Bottles, Ward's DNA Stain (500 mL)
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18
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Microfuge "reaction" tubes
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2
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Gel staining trays
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1
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Spatula ("gel handler")
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30
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Sheets, graph paper
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1
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"Student Study Sheet" copymaster
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1
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"Student Analysis Sheet" copymaster
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1
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Teacher's Manual
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Pre-Laboratory Preparation Procedure
|
(1)
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Prepare TBE Running Buffer (1X) according to the
following directions: to 20 mL TBE running buffer
concentrate (10X) add 180 mL distilled water. This will make a working buffer solution of 1X. This 200 mL quantity is enough for 1 gel.
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(2)
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Cast 0.8% agarose gels. Refer to Section 2.3 in "Ward's
Agarose Electrophoresis Manual" or product literature
supplied with pre-cooked agarose. Each student team
will run 3 sample lanes (lambda DNA EcoR I; lambda
DNA Hind III, lambda DNA EcoR I / Hind III double
digest). Suggested run formats below:
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Student Teams
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(5 students / team)
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No. Gels (6-well)
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No. Gels (12-well)
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1
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1
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1
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2
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1
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1
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3
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2
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1
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4
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2
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1
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5
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3
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2
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6
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3
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2
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(3)
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In this experiment 3 DNA samples will be electrophoresed. It is suggested
that each student team receive their own set of labeled DNA samples
prepared in advance by you.
Prelabel student microfuge "reaction" tubes (set of 3
tubes per student group) as follows:
Tube #1 "lambda DNA EcoR I digest"
Tube #2 "lambda DNA Hind III digest"
Tube #3 "lambda DNA EcoR I / Hind III double digest"
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(4)
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Using a micropipet (see "micropipeting tips"), transfer
EXACTLY 10 uL of each of the above sample types
into corresponding reaction tubes. (Use of a microfuge
tube rack is recommended.) Refreeze any unused
DNA sample.
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(5)
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Heat Tube # 2 at 65deg. C for 5 minutes. Heating helps
band imaging. Note: Use the gray sample container as a "micro water bath."
Place tube #2 into the container and pour water (90deg. C) up to the dividers.
Close the cover. Allow to stand 5 minutes. Average incubation temperature will
be 65deg.C).
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(6)
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Use the enclosed Ward's copymasters ("Student Study" and "Student Analysis") to make worksheet copies. Permission is granted for unlimited copies made for use within any single school building.
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Additional Prep Notes:
|
*
|
You may wish to designate one area of the laboratory as an
"electrophoresis area" where cells and power supplies are connected. Assure
that any wall outlet is properly wired; i.e., that correct polarity exists (use
a circuit tester). Assure that electrical power cords and patch cords are clear
from moisture or wetness.
|
|
*
|
Assure that all students are thoroughly drilled on the correct
procedure regarding making electrical connections and that they are directly
supervised by you.
|
|
*
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Assure that all students wear the following personal protective
equipment: safety goggles, smock or apron (while loading gels and during
electrophoresis), and protective gloves (during staining).
|
Materials for each Team:
|
1
|
Agarose gel 0.8% on gel tray
[per 2 or 4 student teams]
Prepared samples in labeled microfuge "reaction" tubes:
Tube #1 "lambda DNA EcoR I digest"
Tube #2 "lambda DNA Hind III digest"
Tube #3 "lambda DNA EcoR I / Hind III double digest"
Capillary micropipets (10 uL)
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5
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"Student Study Sheets" copymaster
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5
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"Student Analysis Sheets" copymaster
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200
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mL TBE running buffer (1X)
[per 2 or 4 student teams]
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1
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250 mL beaker (for distilled water & running buffer)
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5
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Pair eye goggles
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5
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Aprons/smocks
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5
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Pair protective gloves
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1
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Capillary micropipet bulb assembly
Calculators
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Common Materials:
Ward's power supply
Electro-CellTM
2 Staining trays
1 Bottle, Ward's DNA stain (100 mL per gel)
Distilled water
Spatula "gel handler"
General note: The DNA sample amounts in the labeled microfuge "reaction"
tubes are extremely small. Your teacher will demonstrate the correct procedures
needed to transfer samples from these reaction tubes to the wells on your
gel.
Loading And Running A Gel:
WEAR EYE PROTECTION FOR ALL THESE STEPS
WARNING: The power supply produces high enough voltage to cause severe
electrical shock if handled improperly. For safe operation of this unit, follow
all directions and precautions. Assure that all students are thoroughly drilled
on the procedure regarding the use of this unit, making electrical connections,
and that they are directly supervised by you. We strongly urge you to take the
time to examine all components of your electrophoresis apparatus prior to each
use. The
maintenance inspection should include all cords, plugs, Electro-Cell(TM)
apparatus, and power supply.
Precautions:
Use the same precautions as you would with any other electrical device. This
device produces high enough voltage, although low compared to general
electrophoretic standards, to cause severe electrical shock.
Designate one area of the laboratory as an "electrophoresis area" where cells
and power supplies are connected. Place the power supply near the
Electro-Cell(TM) to which it is to be connected but away from normal everyday
activities.
Do not plug Power Supply into wall receptacle until the safety cover is
positioned on the cell and all other electrical connections are properly made.
This unit uses a 3-wire grounded plug. For safety reasons it should NOT be used
with a 2-wire receptacle with a
conversion plug.
Do not operate in a damp or humid environment since any condensed moisture may
short out electrical components. Assure that the power supply, electrical cord,
and patch cords are clear from wetness or moisture.
Inspect all power cords, patch cords, banana jacks, and plugs for any defects,
such as cracked and dried-out insulation, and loose or wobbly banana jacks or
plugs.
Do not come in personal contact or allow metal or any conductive material to
come in contact with reservoir buffer or the electrophoretic cell while power
supply is on.
The power supply may continue to produce some voltage even when the power has
been turned off. To eliminate any risk associated with this event, follow all
required steps given in the procedure, when disconnecting the electrophoretic
chamber (Electro-Cell(TM)) be sure that the leads do not touch each other, come
in contact with the buffer, or otherwise create any hazardous electrical
condition.
|
(1)
|
On a gel, load the entire sample (10 uL) from each
microfuge tube onto a corresponding gel lane. If more
than one student team is using a gel, keep track of
which well number (from left to right) of a given
sample is placed. Be careful not to puncture the
bottom of the well. Your teacher will demonstrate the
correct procedure.
Lane 1 (Tube #1) "lambda DNA EcoR I digest"
Lane 2 (Tube #2) "lambda DNA Hind III digest"
Lane 3 ( Tube #3) "lambda DNA EcoR I / Hind III
double digest"
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|
(2)
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Place your loaded gel (on the gel tray) in the center
island of the cell. Orient the gel so that the wells are
closest to the BLACK (cathodic) electrode.
|
|
(3)
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Pour approximately 200 mL of TBE running buffer
(1X) into a beaker. Use the beaker to gently pour
running buffer into one buffer compartment of the
cell. As the level reaches the gel, pour running buffer
into the other buffer compartment until it too reaches
the level of the gel. Slowly add enough running buffer
into the compartment nearest the Red (anode)
terminal until the level of buffer is approximately
2 mm ABOVE the top surface of the gel. This type of
gel is called a "submarine." DO NOT OVERFILL the buffer compartments!
|
|
(4)
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Wipe dry the Safety Cover and place it on the
Electrophoretic Cell (Electro-Cell(TM)). Assure that the
Electro-Cell(TM) is not overfilled with buffer. The buffer
level should be approximately 2 mm ABOVE the top
surface of the gel. Wipe off any spills around the
electrophoretic apparatus and power supply before
proceeding to the next step.
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|
(5)
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Make sure that the power supply is unplugged and is
switched off.
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|
(6)
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Assure that the patch cords are completely dry,
including the female plugs, as well as the banana
jacks, on the patch cords and Electro-Cell(TM).
Connect the Red (positive) patch cord to both the Red
electrode terminal on the cell and the Red terminal on
the power supply. Follow the same procedure with
the Black (negative) patch cord.
|
|
(7)
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Plug the power cord into a typical 120V receptacle.
Assure that the receptacle is properly wired; i.e., that
correct polarity exists.
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|
(8)
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Ward's 2-cell and 3-cell power supplies have a preset voltage output. If you are using Ward's 15-5340 power supply, set at 90 V. Refer to "Ward's
Agarose Electrophoresis Manual," section 2.2 for specific information regarding
voltage settings and running times.
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Teacher's Note: Refer to Ward's "Agarose Electreophoresis Manual," Section 2.2 for information regarding voltage settings and running time.
|
|
(9)
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Turn on the power supply. The run light willilluminate signifying that
power is running to the cell.Observe the bubbles that form along the platinum
electrodes.
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(10)
|
Observe the migration of the sample down the gel towards the Red (anodic)
electrode. Turn off the power when the loading dye has run halfway off the
gel.
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(11)
|
Unplug the power supply.
|
|
(12)
|
For safety reasons wait approximately 10 seconds, then first disconnect
the patch cords from the power supply, and afterwards from the
Electro-Cell(TM).
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(13)
|
Carefully notch one end of the gel so that future orientation will be
assured. Lift the gel tray (contain-ing the gel) from the cell and place the
gel in the staining tray. Do this by GENTLY pushing the gel off the tray using
the edge of the "gel handler."
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|
(14)
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WEARING PROTECTIVE GLOVES pour approximately 100 mL of DNA Stain into the
staining tray. Do NOT pour stain directly onto the gel. Cover and label.
Staining will be complete in about 3 hours.
|
|
(15)
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Following staining, carefully decant the used stain.DO NOT allow the gel
to move up against a corner. It MUST remain flat, otherwise, it could break.
Decant the stain directly to a sink drain. Flush with water.
|
|
(16)
|
Add distilled water to the staining tray. Do NOT pour water directly onto
the gel. The tray may be gently rocked to promote destaining. Destain until
bands are distinct, with little background. This will take between
8 and 12 hours, depending upon the degree of agitation used.
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|
(17)
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Wash your hands thoroughly before leaving the lab.
|
|
(18)
|
Observe the gradual destaining of the gel. Destaining is completed when
DNA bands are clearly observed against an almost clear background.
Teacher's note: Use the spatula ("gel handler")
provided in the kit to remove the gel from the staining
tray. Wrap the gel in clear kitchen wrap and store
under refrigeration. (Gel bands will retain the stain
for about one month before they start to fade. Fading
is due to the oxidation of DNA since it [DNA] is not
fixed in the gel matrix.)
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(19)
|
Analyze results. See your Student Analysis Sheet.
|
(Student Analysis Sheet)
Analysis Steps 1-4:
Teacher's notes:
Step 1. Measure the distance of the DNA bands (in mm) from each of the three
sample wells and draw an illustration of the DNA bands in each gel lane.
Note: The diagram of a gel on the left, has an example of a student's drawn
illustration of the DNA bands in one lane.
Step 2. Record migration distance and Rf values for each sample fragment imaged on the gel in the table below. Express fragment size in Kbp (Kilobase pairs)
|
(1)
|
Under a given set of electrophoretic conditions (i.e.,
pH, voltage, time, gel type, concentration, etc.) the
electrophoretic mobility of a DNA fragment molecule
is standard. Thus the length of a given DNA fragment
can be determined by comparing its electrophoretic
mobility on an agarose gel with that of a DNA marker
sample of known lengths. The smaller the DNA
fragment, the faster it will move down the gel during
electrophoresis. Each fragment has (under identical
electrophoretic conditions) a "relative mobility" value
(Rf). Rf can be expressed as:
distance the DNA fragment has migrated
from the origin (gel well)
distance from the origin to the reference
point (end of the gel)
A standard curve is constructed by plotting the Rf
value of each standard DNA fragment versus the
logarithm of its molecular size. The molecular size of
the DNA fragment is the antilog of this number. Both
standard curve plots and published fragment size data
are presented below.
Note: The values presented in the Experimental Data
Table on page 7 were obtained from the gel whose
image data was transferred to the drawing. Student
values will vary, however, they should be close to
published values (Table III) -at least to 2 significant
figures.
|
|
(2)
|
Standard curve plot(s) presented below. Note that
large DNA fragments are not on a line-of-best-fit.
|
Step 3:
Compare your experimental data with the published data in the "Published Data
Table." Fragment base pair values within ( ) denote fragments too small to be
imaged on your gel. Your experimental values should agree with published data,
at least to 2 significant figures. Complete your table with published values
for fragments too small to be imaged.
|
Step 4:
Use Table III to calculate cleavage site position order for each imaged and
non-imaged fragment. Write this value in the [ ] on your table. (Remember that
an RE that cuts linear DNA N times will yield N=1 fragments.)
|
| EcoR I |
Hind III |
EcoR I / Hind III |
|
Tube #1
|
Tube #2
|
Tube #3
|
|
21226 [1]
|
23130 [1]
|
21226 [1]
|
|
7421 [4]
|
9416 [4]
|
5148 [7]
|
|
5804 [5]
|
6557 [7]
|
4973 [11]
|
|
5643 [3]
|
4361 [8]
|
4268 [6]
|
|
4878 [2]
|
2322 [3]
|
2037 [3]
|
|
3530 [6]
|
2037 [2]
|
1894 [2]
|
|
|
(564) [5]
|
1584 [10]
|
|
|
(125) [6]
|
1375 [5]
|
|
|
|
947 [4]
(831) [12]
(564) [8]
(125) [9]
|
[ ] indicates cleavage site position order. (Data from Table III)
( ) indicates fragment sizes too small to be imaged on the gel.
Student data should be expressed in Kilobase pairs (Kbp); thus, a fragment size
of 21,226 bp is expressed as 21.2 Kbp. Subsequent student map plots (Question
1) should agree with published data presented on your maps.
Question One:
Using the data from your Table, construct restriction maps for each of the
following digests:
lambda DNA EcoR I digest
lambda DNA Hind III digest
lambda DNA EcoR I / Hind III double digest
Teacher's note:
Completed maps are presented at the bottom of this page.
Published data is used. See Figure 5 for an example.
The restriction map for the combination lambda DNA digest (EcoR I/Hind III) is
drawn in the following manner: beginning at the left, find the first
restriction site location that occurs in EITHER individual map (it is position
21,226 in the EcoR I digest). Find the NEXT MUTUAL SITE LOCATION (it is 23,120
in the Hind III digest). To calculate the theoretical fragment length between
restriction sites 1 and 2, subtract 21,226 from 23,120, or 1,894. Thus there is
a fragment 1.89Kbp from the first site position to the second site position in
the restriction map of the double digest of lambda DNA using EcoR I/Hind III.
Now, find the third site position (it is 25,157 on Hind III). The distance
between it and the second site fragment is 2.03 Kbp. The data below summarizes
these calculations and also presents ranked fragments by position following
electrophoresis:
|
EcoR I / Hind III Digest
|
Site
No.
|
Fragment
size
|
(calculation)
|
Fragment
Position on Gel
|
|
(1)
|
212261
|
|
(2)
|
1894
|
[23120 - 21226]
|
7
|
|
(3)
|
2037
|
[25157 - 23120]
|
6
|
|
(4)
|
947
|
[26104 - 25157]
|
[10]
|
|
(5)
|
1375
|
[27479 - 26104]
|
9
|
|
(6)
|
4268
|
[31747 - 27479]
|
4
|
|
(7)
|
5148
|
[36895 - 31747]
|
2
|
|
(8)
|
564
|
[37459 - 36895]
|
[12]
|
|
(9)
|
125
|
[37584 - 37459]
|
[13]
|
|
(10)
|
1584
|
[39168 - 37584]
|
8
|
|
(11)
|
4973
|
[44141 - 39168]
|
3
|
|
[12]
|
831
|
[44972 - 44141]
|
[11]
|
|
(13)
|
3530
|
[48502 - 44972]
|
5
|
Expected (theoretical) gel fragment lengths are ranked by position (from
highest nucleotide number to lowest) as they appear on each gel lane. Values
given in [ ], above, denote fragments too small to be imaged on this percent
agarose gel.
Question Two:
From your restriction maps, identify the DNA bands that contain the genes for
head proteins and those that encode for cell lysis.
Teacher's notes:
- EcoR I:
- The DNA fragment length of 21226 bp (fragment 1) contains the genes for head proteins; fragment 6 (3530 bp) contains genes for cell lysis. (Fragment number in decending order as imaged on gel).
- Hind III:
- The DNA fragment length of 23120 bp (fragment 1) contains genes for head proteins; fragment 4 (4361 bp) contains genes for cell lysis. (Fragment number in decending order as imaged on gel.)
- EcoR I/Hind III: (lane 3)
- The DNA fragment length of 21226 bp (fragment 1) contains genes for head proteins; fragment 5 (3530 bp) contains genes for cell lysis. (Fragment number in decending order as imaged on gel.)
The reactions required for genetic analysis at the mlecular level require VERY
small amounts of DNA, reagents, and enzymes. The two most common units of
liquid measurement that are used in these laboratory experiments are the
milliliter (mL) and the microliter (uL).
|
1 mL = 1/1,000 liter
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1,000 mL = 1 liter
|
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1 uL = 1/1,000,000 liter
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1,000,000 uL = 1 liter
|
Use of Micropipets:
Digital micropipet
| There are three important DON'TS when using a digital micropipet: |
|
(1)
|
DON'T attempt to use the pipet without a tip in place. This can ruin the
precision piston that determines the volume of the fluid.
|
|
(2)
|
DON'T lay down a pipet that has a filled tip. Fluid in the tip could
run back into the pipet and ruin the precision piston.
|
|
(3)
|
DON'T let the delivery button (control button) snap back after
withdrawing or delivering the fluid.
|
To use a digital pipet:
|
(1)
|
Rotate the delivery button to the desired volume.
|
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(2)
|
Push the pipet, firmly, into the proper size micropipet tip.
|
|
(3)
|
While withdrawing or expelling liquid, hold the vessel and pipet at
nearly eye level. It is important to watch while you pipet.
|
|
(4)
|
Hold the pipet close to the vertical while pipeting.
|
To withdraw a sample:
|
(1)
|
Depress the button to the first stop and hold the button in that
position. Dip the tip of the pipet into the fluid to be pipeted and slowly
release the button to draw the fluid into the tip.
|
|
(2)
|
Slide the tip of the pipet along the side wall of the reagent tube to knock
off excess fluid that might have remained on the outside of the tip.
|
To expel a sample into a reagent tube:
|
(1)
|
Touch the pipet tip to the in side wall of the reaction tube (microfuge
tube) into which the sample will go. This will help create a capillary effect
which should help draw the fluid out of the pipet tip.
|
|
(2)
|
To expel the sample, slowly depress the button to the first stop. Wait a
second or two and then press on to the second stop to blow out the last bit
of fluid.
Continue to hold the button in the second position as the pipet tip is
withdrawn from the tube.
|
|
(3)
|
To eject the tip (if the pipet has this feature), press the ejector
button down while the pipet is held over a beaker which has been designated
as the proper receptacle for used tips.
|
To prevent contamination of the reagents used in the
laboratory, follow these guidelines:
- Always add appropriate amounts of a single reagent sequentially to all reaction (microcentrifuge) tubes.
- Release each reagent drop onto a NEW LOCATION on the inside wall of the reaction tube. In this way you can continue to use the same tip to pipet a number of samples of a single reagent into different reaction tubes.
- Use a fresh pipet tip for each new reagent you pipet.
Microcapillary pipet
Capillary micropipets are capillary tubes that are marked to accept a specific
microquantity of fluid. Usually they employ a bulb-type plunger, or a thin
metal rod for suction. If due care is used, they are an accurate and economical
way to apply samples for use in electrophoresis.
(Bulb Type)
|
(1)
|
Insert the pipet through the hole pierced in the amber rubber cap.
|
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(2)
|
Hold the black rubber bulb between the thumb and third finger.
|
|
(3)
|
Fill pipet by capillary attraction.
|
|
(4)
|
To expel place index finger over the hole in the black rubber bulb and
squeeze.
|
(Piston Type)
|
(1)
|
Insert the metal piston (wire) all the way into the capillary tube.
|
|
(2)
|
Dip the tip of the capillary tube into the fluid, pulling back on the
wire piston to fill tube. Draw up to the line.
|
|
(3)
|
To expel, push down on the wire piston.
|
To prevent contamination of the reagents used in the
laboratory, follow these guidelines:
- Always add appropriate amounts of a single reagent sequentially to all reaction (microcentrifuge) tubes.
- Release each reagent drop onto a NEW LOCATION on the inside wall of the reaction tube. In this way you can continue to use the same tip to pipet a number of samples of a single reagent into different reaction tubes.
- Use a fresh pipet tip for each new reagent you pipet.
Restriction Mapping of Lambda DNA:
Materials in Ward's GEL-Kit:
DNA samples:
|
2
|
lambda DNA Hind III
TO ORDER --> [Vial, 10 ug]
|
85W1331
|
|
2
|
lambda DNA EcoR I
TO ORDER --> [Vial, 10 ug]
|
85W1332
|
|
2
|
lambda DNA Hind III/ EcoR I
TO ORDER --> [Vial, 10 ug]
|
85W1337
|
Reagents:
|
2
|
Bottles, Ward's DNA stain
TO ORDER --> [Btl, 500 mL]
|
38W9012
|
|
1
|
Bottles, 0.8% prepared agarose
TO ORDER --> (Btl, 200 mL)
[Pkg/6]
|
88W1207
|
|
1
|
Bottle, TBE (10X) Running buffer
TO ORDER --> (Btl, 250 mL) [each]
|
37W0620
|
Other Materials:
|
2
|
Gel staining trays
TO ORDER --> [each]
|
18W0031
|
|
1
|
Spatula ("gel handler")
TO ORDER --> [each]
|
15W9853
|
|
1
|
Pack, graph paper (40 sheets)
TO ORDER --> [2 x 20]
|
15W3835
|
|
1
|
"Student Analysis Sheet" copymaster
|
|
1
|
"Student Study Sheet" copymaster
|
|
1
|
Teacher's Manual
|
|
18
|
Microfuge tubes
TO ORDER --> [Pkg/500]
|
18W1361
|
Micropipets:
Eppendorf (precision measurement device)
TO ORDER --> [each]
|
15W2084
|
Eppendorf micropipet tips (Gel-Loader)
TO ORDER --> [Pkg/200]
|
15W2089
|
Capillary micropipet (5/10 uL)
TO ORDER --> [Pkg/250 w/2 plungers]
|
15W2094
|
Microcap micropipet bulb assembly
TO ORDER --> (For 15W2090) [Pkg/5]
|
15W2092
|
Microcap micropipet (10 ul)
TO ORDER --> [Pkg/100]
|
15W2091
|
Other Materials:
Strapping tape
TO ORDER --> [each]
|
15W1980
|
Safety goggles
TO ORDER --> [each]
|
15W3119
|
Apron (disposable)
TO ORDER --> [Pkg/100]
|
15W1050
|
Gloves (disposable)
TO ORDER --> (sm/med) [Box/100]
TO ORDER --> (lg) [Box/100]
|
15W1073
15W1074
|
Gloves (thermal protection)
TO ORDER --> [each pair]
|
15W1095
|
Electro-Cell(TM)
TO ORDER --> [each]
|
36W5110
|
Ward's Power Supply
TO ORDER --> [each]
|
36W5112
|
Calculator
TO ORDER --> [each]
|
27W2055
|
Gel casting tray
TO ORDER --> [each]
|
36W5113
|
Gel comb (6-well)
TO ORDER --> [each]
|
36W5114
|
Gel comb (12-well)
TO ORDER --> [each]
|
36W5115
|
Rulers (6" Metric/English)
TO ORDER --> [each]
|
14W0810
|
For Technical Assistance, Call Toll Free:
(800) 962-2660
Ward's Natural Science Establishment, Inc.
Copyright © 1990
AE Partners Collection Index
