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CHEF Genomic DNA Plug Kits
Instruction Manual
Catalog Numbers
170-3591
170-3592
170-3593
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
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Table of Contents
Section 1 Introduction......................................................1
1.1 Kit Components ..........................................................2
Section 2 Preparation of Agarose Embedded
Section 3 Preparation of Agarose Embedded
Section 4 Preparation of Agarose
Section 5 Restriction Enzyme Digestion of Plugs........11
Section 6 References ......................................................12
Section 7 Additional Reagents for Pulsed Field
Section 8 Instruments for Pulsed Field
Section 9 Appendix ........................................................14
Mammalian DNA ............................................3
Bacterial DNA..................................................5
Embedded Yeast DNA ....................................8
Electrophoresis ..............................................13
Electrophoresis ..............................................13
9.1 Solutions....................................................................14
9.2 Agarose Concentrations ............................................15
9.3 Hemocytometer Usage ..............................................16
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Section 1
Introduction
Pulsed-Field Gel Electrophoresis (PFGE) allows the
separation of DNA ranging in size from a few kilobase pairs
to 10 megabase pairs. Because of the large size of these
molecules, simple pipetting mechanically shears the DNA
resulting in unacceptable quality for PFGE separations. This
has necessitated procedures for lysis of whole cells embedded
in agarose, allowing purification of chromosome-sized DNA
without shearing.
The most important and difficult task in preparing cells
for embedding in agarose is to obtain the proper cell
concentration. Although optical density is frequently used to
determine cell concentration, it is not reliable. Different
strains, plasmid content, and growth media all contribute to
the actual cell number achieved for a particular optical
density. Variation in cell number will cause the amount of
DNA per agarose plug to vary, leading to over- and or underloading of the sample. To eliminate the need to generate a
growth curve for each strain, a hemocytometer is the most
reproducible method for achieving the proper cell
concentration for different types of cells, bacteria, yeast, and
fungi. Detailed instructions for the use of a hemocytometer
are given in the Appendix.
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1.1 Kit Components
The CHEF Genomic DNA Plug Kits are designed to
produce 100 sample plugs of agarose embedded DNA with
the quality necessary for PFGE separations. All kits consist
of a core module, which contains all the buffers and
proteinase solutions. This core module is the only component
supplied with the mammalian DNA kit. Other kits contain
additional modules with lysis buffers specific to that cell
type. For example, the bacterial kit contains lysozyme and its
reaction buffer necessary to digest the outer cell membrane of
lysozyme-sensitive bacteria; the yeast module contains
lyticase and its reaction buffer necessary to digest the cell
walls of most yeast. Each of the disposable plug molds
provided contains 50 wells which are 1.5 mm thick and 5 mm
wide and hold 85 µ l of volume. It is also possible to use the
10 well reusable sample plug mold (170-3622). Each of the
wells in the reusable mold are 1 cm wide and 1.5 cm thick
and hold 300 µl of volume.
Catalog
Number Product Description
170-3591 CHEF Mammalian Genomic DNA Plug Kit, contains
170-3592 CHEF Bacterial Genomic DNA Plug Kit, contains
Cell Suspension Buffer, 12 ml; >600 U/ml Proteinase K,
1.3 ml; Proteinase K Reaction Buffer, 30ml;
2% CleanCut
50 well disposable plug mold, 2; Screened Cap, 1
Cell Suspension Buffer, 12 ml; >600 U/ml Proteinase K,
1.3 ml; Proteinase K Reaction Buffer, 30 ml;
2% CleanCut Agarose, 12 ml; 10x Wash Buffer, 60 ml;
25 mg/ml Lysozyme, 1.6 ml; Lysozyme Buffer, 30 ml,
50 well disposable plug mold, 2; Screened Cap, 1
™
Agarose, 12 ml; 10x Wash Buffer, 60 ml,
170-3593 CHEF Yeast Genomic DNA Plug Kit, contains Cell
Suspension Buffer, 12 ml; >600 U/ml Proteinase K,
1.3 ml; Proteinase K Reaction Buffer, 30 ml;
2% CleanCut Agarose, 12 ml; 10x Wash Buffer, 60 ml;
5000 U/ml Lyticase, 1.6 ml; Lyticase Buffer, 30 ml,
50 well disposable plug mold, 2; Screened Cap, 1
Section 2
Preparation of Agarose Embedded
Mammalian DNA
Reagents and Equipment Needed
Sterile transfer pipettes
50 °C water bath
PMSF stock solution see Appendix
Hemocytometer see Appendix
Microscope
2, 5, 50 ml sterile plastic tubes
1. Prepare a cell suspension in isotonic saline or tissue
culture medium without fetal bovine serum. Count the
cells and remove 5 x 10
plugs to be made (use 100 µ l/plug for disposable mold or
300 µ l/plug for reusable mold) and place on ice.
See Appendix for hemocytometer usage.
2. Melt the 2% CleanCut agarose solution using a
microwave and equilibrate the solution to 50 °C in a
water bath.
7
cells for each ml of agarose
2
3
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3. Calculate the amount of Cell Suspension Buffer
and CleanCut agarose necessary (see Section 9.2). For a
final concentration of 0.75% agarose use 0.63 ml of Cell
Suspension Buffer per ml of agarose plugs. Use 0.37 ml
of 2% CleanCut agarose per ml of agarose plugs.
4. Centrifuge the cell suspension at 1,000 x g for 5 minutes
at 4 °C. Resuspend the cells in the volume of Cell
Suspension Buffer calculated above and equilibrate the
cell suspension to 50 °C.
5. Combine the calculated volume of 2% CleanCut agarose
with the cell suspension and mix gently, but thoroughly.
Keeping the cell/agarose mixture at 50 °C, transfer the
mixture to plug molds using sterile transfer pipettes
(Bio-Rad's disposable transfer pipettes, catalog 223-9524,
are recommended). Allow the agarose to solidify. This
step can be expedited by placing the molds at 4 °C for
10–15 minutes, which also adds strength to the agarose
for removal from the mold.
6. Using a 50 ml conical centrifuge tube, add 100 µ l of
Proteinase K stock to 2.5 ml of Proteinase K Reaction
Buffer for each ml of agarose plugs. Push the solidified
agarose plugs into the 50 ml centrifuge tube containing
the Proteinase K solution. Incubate the plugs overnight at
50 °C without agitation.
Note: Various cell lines have been incubated up to 4 days
in Proteinase K without detrimental effects to the quality
of DNA.
Note: For processing a few plugs, use a 2 or 5 ml tube.
7. Wash the plugs four times in 1x Wash Buffer, 1 hour each
at room temperature with gentle agitation (for each plug,
use 1 ml of 1x Wash Buffer). Prepare the Wash Buffer by
diluting the 10x stock (1:10) with sterile ddH
plugs are to be used in subsequent enzyme reactions, it is
advisable to wash the plugs in 1 mM PMSF during the
second or third wash to inactivate residual Proteinase K.
See Appendix for PMSF stock solution.
Note: For washing a few plugs, use a 2 or 5 ml sterile tube.
8. Store the plugs at 4 °C in 1x Wash Buffer. The plugs
should be stable for 3 months.
O. If the
2
Section 3
Preparation of Agarose Embedded
Bacterial DNA
Reagents and Equipment Needed
Sterile transfer pipettes
50 °C water bath
Grams Crystal Violet (Difco)
Microscope
PMSF stock solution see Appendix
Hemocytometer see Appendix
2, 5, 50 ml sterile plastic tubes
1. Inoculate a bacterial culture into 50 ml of LB Broth or
appropriate media and grow with agitation to an O.D.
of 0.8–1.0 at the appropriate temperature. See appendix
for LB Broth.
600
4
5
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2. When the desired O.D.
is reached, add chloramphenicol
600
to a final concentration of 180 µg/ml and continue
incubation up to 1 hour while performing step 3.
Note: Chloramphenicol is used to synchronize ongoing
rounds of chromosomal replication and inhibit further
rounds of replication. This step is optional, but regions near
the replication terminus might be under-represented. In
addition, chloramphenicol will alter the morphology of the
cells over time causing the appearance of a mixed culture;
therefore proceed as quickly as possible with step 3.
3. Make a twenty-fold dilution of the above bacterial
suspension using 1 ml bacteria, 1 ml Gram Crystal
Violet, and 18 ml saline or PBS. Place a small amount of
the bacterial suspension on a hemocytometer and count at
400x power. See Appendix on hemocytometer usage.
4. Melt the 2% CleanCut agarose solution using a
microwave or hot water bath and equilibrate the solution
to 50 °C in a water bath.
5. Calculate the amount of Cell Suspension Buffer and
CleanCut agarose necessary (see Section 9.2). For a final
concentration of 1% agarose use 0.5 ml of Cell Suspension
Buffer per ml of agarose plugs (use 100 µ l/plug for
disposable mold or 300 µ l/plug for reusable mold). Use
0.5 ml of 2% CleanCut agarose per ml of agarose plugs.
6. Remove 5 x 10
8
cells for each ml of agarose plugs to be
made. Centrifuge for 3 minutes in a microcentrifuge. If the
volume is too large, spin at 10,000 x g for 5 min at 4 °C in
an appropriate size tube. Resuspend the cells in the volume
of Cell Suspension Buffer calculated above and equilibrate
the cell suspension to 50 °C.
7. Combine the calculated volume of 2% CleanCut agarose
with the cell suspension and mix gently, but thoroughly.
Keeping the cell/agarose mixture at 50 °C, transfer the
mixture to plug molds using sterile transfer pipettes
(Bio-Rad's disposable transfer pipettes, catalog 223-9524,
are recommended). Allow the agarose to solidify. This
step can be expedited by placing the molds at 4 °C for
10–15 minutes, which also adds strength to the agarose
for removal from the mold.
8. Push the solidified agarose plugs into a 50 ml conical
centrifuge tube containing lysozyme solution. Prepare
lysozyme solution by adding 100 µ l of Lysozyme stock
to 2.5 ml of Lysozyme Buffer for each 1 ml of agarose
plugs. Incubate the plugs for 2 hours at 37 °C.
Note: For processing a few plugs, use a 2 or 5 ml tube.
9. Remove the lysozyme solution and rinse the plugs with
sterile water. Add 2.5 ml of Proteinase K Reaction Buffer
for each ml of agarose plugs, followed by 100 µ l of
Proteinase K stock. Incubate the plugs overnight at 50 °C
without agitation.
Note: Various cell lines have been incubated up to 4 days
in Proteinase K without detrimental effects to the quality
of DNA.
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10. Wash the plugs four times in 1x Wash Buffer, 1 hour each
at room temperature with gentle agitation (for each plug,
use 1 ml of 1x Wash Buffer). Prepare the Wash Buffer by
diluting the 10x stock (1:10) with sterile ddH
O. If the
2
plugs are to be used in subsequent enzyme reactions, it is
advisable to wash the plugs in 1 mM PMSF during the
second or third wash to inactivate residual Proteinase K.
See Appendix for PMSF stock solution.
Note: For washing a few plugs, use a 2 or 5 ml sterile tube.
11. Store the plugs at 4 °C in 1x Wash Buffer. The plugs
should be stable for 3 months.
Section 4
Preparation of Agarose Embedded
Yeast DNA
Reagents and Equipment Needed
50 mM EDTA, pH 8
Sterile transfer pipettes
50 °C water bath
Microscope
PMSF stock solution see Appendix
Hemocytometer see Appendix
2, 5, 50 ml sterile plastic tubes
1. Inoculate a single colony into 50 to 100 ml YPD broth or
appropriate media. Grow with agitation to an O.D.
600
>1.0 at the appropriate temperature for your strain.
See Appendix for YPD broth.
2. When the desired O.D.
is reached, centrifuge the cells
600
at 5,000 x g, 10 min, 4 °C. Pour off the supernatant and
resuspend in 10 ml cold 50 mM EDTA, pH 8.
3. Determine the cell concentration by adding 10 µl of cells
to 990 µ l of water. Place the yeast suspension on a
hemocytometer and count at 400x power. See Appendix
on hemocytometer usage.
4. Heat the 2% CleanCut agarose solution using a
microwave and equilibrate the solution to 50 °C in a
water bath.
5. Calculate the amount of Cell Suspension Buffer and
CleanCut agarose necessary (see Section 9.2). For a
final concentration of 0.75% agarose use 0.63 ml of
Cell Suspension Buffer per ml of agarose plugs (use
100 µ l/plug for disposable mold or 300 µl/plug for
reusable mold). Use 0.37 ml of 2% CleanCut agarose
per ml of agarose plugs to be made.
6. Remove 6 x 10
8
cells for each ml of plugs to be made.
Centrifuge in a microfuge for 3 minutes if volumes are
small, otherwise centrifuge the cells at 5,000 x g, for
10 minutes at 4 °C. Resuspend the cells in the volume of
Cell Suspension Buffer calculated above and equilibrate
to 50 °C.
of
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7. Just prior to mixing the cells with agarose, add 30 µ l of
the Lyticase stock, for each ml of plugs to be made, to the
cell suspension.
Note: It is recommended that Lyticase be added immediately prior to imbedding the cells in agarose. It has been
found that certain strains do not give acceptable DNA
when Lyticase is allowed to diffuse into the agarose plug.
8. Combine the calculated volume of 2% CleanCut agarose
with the cell suspension and mix gently, but thoroughly.
Keeping the cell/agarose mixture at 50 °C, transfer the
mixture to plug molds using sterile transfer pipettes
(Bio-Rad's disposable transfer pipettes, catalog 223-9524,
are recommended). Allow the agarose to solidify. This
step can be expedited by placing the molds at 4 °C for
10–15 minutes, which also adds strength to the agarose
for removal from the mold.
9. Push the solidified agarose plugs into a 50 ml conical
centrifuge tube containing Lyticase solution. Prepare
Lyticase solution by adding 85 µ l of Lyticase stock to
2.5 ml of Lyticase Buffer for each 1 ml of plugs. Incubate
the plugs for 2 hours at 37 °C.
Note: For processing a few plugs, use a 2 or 5 ml tube.
10. Remove the lyticase solution and rinse the plugs with
sterile water. Add 2.5 ml of Proteinase K Reaction Buffer
for each ml of agarose plugs, followed by 100 µl of
Proteinase K stock. Incubate the plugs overnight at 50 °C
without agitation.
Note: Various cell lines have been incubated up to 4 days
in Proteinase K without detrimental effects to the quality
of DNA.
11. Wash the plugs four times in 1x Wash Buffer, 1 hour each
at room temperature with gentle agitation (for each plug,
use 1 ml of 1x Wash Buffer). Prepare the Wash Buffer by
diluting the 10x stock (1:10) with sterile ddH
plugs are to be used in subsequent enzyme reactions, it is
advisable to wash the plugs in 1 mM PMSF during the
second or third wash to inactivate residual Proteinase K.
See Appendix for PMSF stock solution.
Note: For washing a few plugs, use a 2 or 5 ml sterile tube.
12. Store the plugs at 4 °C. The plugs should be stable for
3 months.
O. If the
2
Section 5
Restriction Enzyme
Digestion of Plugs
1. Place one plug per digest in a sterile 1.5 ml microcentrifuge
tube. Wash once for 1 hour in 1 ml 0.1x Wash Buffer
(1:100 dilution of 10x stock Wash Buffer) Use 1 ml 0.1x
Wash Buffer per plug. Decant and resuspend in a sufficient
amount of fresh 0.1x Wash Buffer to cover the plugs. This
last wash reduces the EDTA concentration, allowing faster
buffer equilibration with restriction enzyme buffers.
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2. Aspirate the Wash Buffer and add 1 ml of the appropriate
1x restriction enzyme buffer for about 1 hour with gentle
agitation at room temperature. Aspirate off the buffer and
add 0.3 ml of fresh 1x enzyme buffer. Add the restriction
enzyme (30–50 U per 100 µ l plug) and incubate overnight
at the appropriate temperature.
3. After overnight digestion, remove the buffer and incubate
in 1 ml of 1x Wash Buffer for approximately 30 minutes
with gentle agitation.
4. Optional: Remove the Wash Buffer and equilibrate the
plug in the appropriate concentration of gel running
buffer, i.e. 0.5x TBE, 1.0x TAE, etc.
5. Load
1
⁄4 to 1⁄3 of a plug per well (this is approximately 50 µg
DNA) and adjust the volume if necessary on subsequent
gels. In addition, always load appropriate size standards.
Section 7
Additional Reagents for
Pulsed Field Electrophoresis
Catalog
Number Product Description
162-0137 Pulsed Field Certified Agarose, 100 g
162-0135 Chromosomal Grade Agarose, 25 g
170-3594 CleanCut Agarose, 2%, 12 ml
170-3624 DNA Size Standards, 5 kb ladder
170-3605 DNA Size Standards, Yeast chromosomal
170-3633 DNA Size Standards,
170-3635 DNA Size Standards, lambda ladder
162-0196 Zeta-Probe
161-0733 10x TBE Buffers, 1 liter
®
GT Nylon Membrane, roll, 30 cm x 3.3 m
S. pombe
chromosomes
Section 6
References
1. Smith, C. L., Klco, S. R. and Cantor, C. R., Genome Analysis,
Chapter 3, K. Davis ed., IRL Press Ltd., Oxford, England (1988).
2. Smith, C. L. and Cantor, C. R., Methods in Enzymology, 155,
449–467 (1988).
12
Section 8
Instruments for Pulsed Field
Electrophoresis
Catalog
Number Product Description
®
170-3612 CHEF-DR
170-3695 CHEF-DR III System
170-3670 CHEF Mapper®XA Chiller System
170-3654 Cooling Module
165-5031 GS GeneLinker®UV Chamber
II System
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Section 9
Appendix
9.1 Solutions
PMSF, 100 mM Add 0.174 g to 10 ml of 100%
(phenylmethanesulfonyl isopropanol. Store at room temfluoride) perature.
A stock solution of 100 mM in
100% isopropanol is stable at
room temp for 1 year. PMSF is
not stable in aqueous solutions.
Activity lasts approximately 30 to
50 min at room temperature at pH
7.5-8.
LB Per liter:
10 g Bacto Tryptone
5 g Bacto Yeast Extract
10 g NaCl
pH 7
9.2 Agarose Concentrations
Various agarose plug percentages are reported in the
literature. The most common final concentration is 0.5%, but
the softness of these plugs makes them difficult to keep intact
and handle. We have found that 0.75% to 1% agarose greatly
enhances the strength of the plug without interfering with the
migration of the DNA out of the plug. These kits contain a 2%
CleanCut agarose solution, which when mixed with differing
amounts of Cell Suspension Buffer, will give agarose
concentrations ranging from 0.5 to 1%. This allows the option
of using a different final concentration of agarose if desired.
Desired Agarose
Concentration
1% (0.5)vol. = ____
CleanCut Agarose
2%*
Cell Suspension
Buffer*
(0.5)vol. = ____
YPD Per liter:
20 g Bacto Peptone
10 g Bacto Yeast Extract
20 g dextrose (glucose)
14
0.75%
0.5%
* Vol. is the total volume of plugs to be made at 75 µl/plug, i.e. for 5 ml
of plugs at 0.75% final agarose concentration, add (0.375)5 ml = 1.9 ml
of 2% agarose solution to (0.625)5 ml = 3.4 ml of cell suspension.
(0.375)vol. = ____
(0.25)vol. = ____
15
(0.625)vol. = ____
(0.75)vol. = ____
Page 11
Cells Counted
Number of Center Squares
= Average Cells per Square
9.3 Hemocytometer Usage
A hemocytometer is usually divided into nine large
squares (Figure 1). Each large square is 1 x 10
3
mm
, two such squares are shown the figure with darkened
borders (A&B). The large circle around the center square (B)
represents your field of view at 100x power (10x objective
lens, 10x eye piece). The center square is subdivided into 25
smaller squares. The smaller circle in the center square (C)
represents your field of view at 400x power (40x objective
lens, 10x eye piece). These 25 center squares are further
divided into 16 squares.
-4cm2
or 0.1
A. Mammalian or Tissue Culture Cells:
Because of the large size, tissue culture cells can be
counted at 100x power. Count 10 of the large squares, five on
each side of the hemocytometer. Determine the average cells
per square using the equations below:
Average Cells per square × Dilution Factor × 10
4
= Cells per ml.
Use the following ratio to determine how many ml of cell
suspension to use to achieve the desired cell concentration for
the plugs.
5 x 107 cells desired
actual cell concentration
× ml of plugs to be made = ml of cell suspension
to use.
Fig. 1. Hemocytometer grid.
B
C
A
16
For Example: 500 cells in 10 squares = average of
50 cells /square x 5 (dilution factor) x 10
ml. For 1 ml of plugs you need 1 ml x (5 x 10
concentration divided by 2.5 x 10
4
= 2.5 x 106cells per
7
6
actual cells concentration =
) cells final
20 ml of cell suspension is required to make 1 ml of agarose
plugs.
B. Bacteria and Yeast Cells:
Count five to ten of the 25 center squares, at 400x power,
to get a representative sample of your cell suspension. You
should have approximately 25 to 75 cells per square. The
cells should be relatively free of clumps. Bacteria which
naturally chain or grow in clusters are relatively easy to count
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Bio-Rad
Laboratories
and do not have to be dispersed by chemical or enzymatic
n
.
methods. The Grams Crystal Violet aids in the visualization
of bacteria.
Use the equations below to determine the cell
concentration:
Cells Counted
Number of Squares
Average Cells per square × 25 Squares × Dilution Factor × 10
desired cell concentration
actual cell concentration
= Average Cells per Square
× ml of plugs to be made = ml of cell suspensio
4
= Cells per ml
to use
For example: 300 bacteria in 5 squares = average of
60 bacteria/square x 25 (squares) x 20 (dilution factor, yeast
use 100 for dilution factor) x 10
For 5 ml of plugs you need 5 ml x (5 x 10
concentration ÷ (3 x 10
8
) actual cells concentration = 8.33 ml
4
= 3 x 108bacteria per ml.
8
) cells final
of cell suspension is required.
8
For yeast use 6 x 10
cells per ml for the final
concentration (see page 9).
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LIT510 Rev E
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