Bio-Rad Criterion TBE-Urea Precast Gels User Manual
Criterion™ Precast Gels
Instruction Manual and Application Guide
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Criterion™ TGX Stain-Free™ and Criterion Stain Free™ precast gels are covered by U.S. Patent No. 7,569,130.
Purchase of Criterion™ XT Bis-Tris gels, XT MOPS running buffer, XT MES running buffer, XT MOPS buffer kit, and XT MES buffer
kit is accompanied by a limited license under U.S. patents 6,143,154; 6,096,182; 6,059,948; 5,578,180; 5,922,185; 6,162,338;
and 6,783,651 and corresponding foreign patents.
Copyright © 2011 by Bio-Rad Laboratories. All rights reserved.
Contents
Chapter 1: Criterion™ Precast Gels................................................... 1
1.1 Introduction ................................................................... 1
1.2 Gel Formulations ............................................................... 2
1.3 Comb Configurations ........................................................... 2
1.4 Specifications ................................................................. 2
1.5 Storage Conditions ............................................................ 3
1.6 Important Notes ............................................................... 3
Chapter 2: Setup and Basic Operation ............................................... 4
2.1 Workflow Overview ............................................................. 4
2.2 Required Materials ............................................................. 5
2.3 Setting Up and Running Criterion Gels in the Criterion Cell ............................... 5
2.4 Removing the Gel .............................................................. 5
Chapter 3: SDS-PAGE............................................................. 7
3.1 Introduction ................................................................... 7
3.2 Criterion Gel Selection Guide for SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2.1 Criterion™ TGX™ and Criterion™ TGX Stain-Free™ Gels .............................. 8
3.2.2 Criterion Tris-HCl and Criterion Stain Free™ Gels................................... 9
3.2.3 Criterion™ XT Bis-Tris Gels .................................................. 10
3.2.4 Criterion XT Tris-Acetate Gels ................................................ 11
3.3 SDS-PAGE Buffers ............................................................ 12
3.3.1 Running Buffers........................................................... 12
3.3.2 Sample Buffers ........................................................... 12
3.4 Sample Preparation............................................................ 12
3.5 Running Conditions ............................................................ 13
Chapter 4: Native PAGE .......................................................... 14
4.1 Introduction .................................................................. 14
4.2 Criterion Gel Selection Guide for Native PAGE ....................................... 14
4.2.1 Criterion TGX and Criterion TGX Stain-Free Gels.................................. 14
4.2.2 Criterion Tris-HCl and Criterion Stain Free Gels ................................... 15
4.2.3 Criterion XT Tris-Acetate Gels ................................................ 15
4.3 Native PAGE Buffers ........................................................... 15
4.4 Sample Preparation............................................................ 16
4.5 Running Conditions ............................................................ 16
Chapter 5: Criterion Stain Free System .............................................. 17
5.1 Introduction .................................................................. 17
5.2 Criterion Stain Free Workflow .................................................... 18
5.3 Electrophoresis with Criterion TGX Stain-Free Gels .................................... 18
5.4 Using the Gel Doc™ EZ Imager .................................................. 18
Chapter 6: Peptide Analysis ....................................................... 19
6.1 Introduction .................................................................. 19
6.2 Criterion Tris-Tricine/Peptide Gels ................................................ 19
6.2.1 Gel Composition .......................................................... 19
6.2.2 Gel Selection Guide........................................................ 19
6.3 Tris-Tricine/Peptide Buffers ...................................................... 19
6.4 Sample Preparation............................................................ 20
6.5 Running Conditions ............................................................ 20
Chapter 7: Isoelectric Focusing (IEF)................................................ 21
7.1 Introduction .................................................................. 21
7.2 Criterion IEF Gels ............................................................. 21
7.2.1 Gel Composition .......................................................... 21
7.2.2 Gel Selection Guide........................................................ 21
7.3 IEF Buffers................................................................... 21
7.4 Sample Preparation............................................................ 22
7.5 Running Conditions ............................................................ 22
Chapter 8: Protease Analysis by Zymogram PAGE .................................... 23
8.1 Introduction .................................................................. 23
8.2 Criterion Zymogram Gels........................................................ 23
8.2.1 Gel Composition .......................................................... 23
8.2.2 Gel Selection Guide........................................................ 23
8.3 Zymogram Buffers............................................................. 23
8.4 Sample Preparation............................................................ 24
8.5 Running Conditions ............................................................ 24
Chapter 9: Nondenaturing Nucleic Acid PAGE ........................................ 25
9.1 Introduction .................................................................. 25
9.2 Criterion TBE Gels............................................................. 25
9.2.1 Gel Composition .......................................................... 25
9.2.2 Gel Selection Guide........................................................ 25
9.3 Nondenaturing Nucleic Acid PAGE Buffers .......................................... 25
9.4 Sample Preparation............................................................ 26
9.5 Running Conditions ............................................................ 26
Chapter 10: Denaturing Nucleic Acid PAGE .......................................... 27
10.1 Introduction ................................................................. 27
10.2 Criterion TBE-Urea Gels ....................................................... 27
10.2.1 Gel Composition ......................................................... 27
10.2.2 Gel Selection Guide....................................................... 27
10.3 TBE-Urea Buffers ............................................................ 27
10.4 Sample Preparation........................................................... 27
10.5 Running Conditions ........................................................... 28
Chapter 11: 2-D Electrophoresis ................................................... 29
11.1 Introduction ................................................................. 29
11.2 Equilibration................................................................. 29
11.3 Agarose Overlay ............................................................. 29
11.4 Second-Dimension Electrophoresis............................................... 29
Chapter 12: Detection............................................................ 30
12.1 SDS-PAGE and Native PAGE Detection ........................................... 30
12.2 Peptide Gel Staining .......................................................... 31
12.3 IEF Gel Staining.............................................................. 31
12.4 Zymogram Gel Staining........................................................ 32
12.5 TBE Gel Staining ............................................................ 32
12.6 TBE-Urea Gel Staining ....................................................... 32
Chapter 13: Blotting ............................................................. 33
13.1 Introduction ................................................................. 33
13.2 Transfer.................................................................... 33
13.2.1 Transfer Buffers .......................................................... 33
13.2.2 Wet Transfer Using the Criterion Blotter ....................................... 33
13.2.3 Transfer Using the Trans-Blot® Turbo™ System.................................. 34
13.2.4 Semi-Dry Transfer Using the Trans-Blot® SD Cell ................................ 36
13.3 Total Protein Blot Stains ....................................................... 36
13.4 Immunodetection ............................................................ 37
Chapter 14: Troubleshooting ...................................................... 38
Appendix A: Quick Start Guide .................................................... 40
Appendix B: Buffers ............................................................. 42
Appendix C: Related Literature .................................................... 45
Appendix D: Ordering Information .................................................. 46
1
Criterion™ Precast Gels
1.1 Introduction
Criterion precast gels are an effective system for performing polyacrylamide gel electrophoresis (PAGE).
These 13.3 x 8.7 cm gels are wider and longer than traditional mini format gels, and their innovative,
easy-to-use design produces excellent resolution while accommodating more samples per gel.
Designed for use with the Criterion family of vertical electrophoresis cells, which includes the Criterion
(2-gel capacity) and Criterion™ Dodeca™ (12-gel capacity) cells, Criterion precast gels allow separation of
more samples than mini format gels and provide significant cost and time savings. Some of the unique
features provided are:
n
Integrated buffer chamber that eliminates buffer leaks
n
Capacity for up to 26 samples per gel
n
Compatibility with multichannel pipets (12+2 and 26-well)
n
Outlined and numbered wells that simplify sample loading and identification
n
Patented1 J-foot design that eliminates post-run gel processing steps and improves gel
drying and blotting results
n
Criterion™ TGX Stain-Free™ formulations for rapid gel imaging without staining
Integrated buffer
chamber
Gel type, expiration
date, and catalog
and lot numbers
Outlined and
numbered wells
1
U.S. patent 6,093,301.
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Criterion Precast Gels
1.2 Gel Formulations
Criterion precast gels are available in a range of formulations for virtually every electrophoresis
application (Table 1.1). All Criterion gels are composed of polyacrylamide with a bisacrylamide
crosslinker, and they are available in a selection of single percentages and gradients.
Table 1.1. Criterion precast gel formulations.
Application Gel Formulation Sample Buffer Running Buffer
SDS-PAGE Criterion Tris-HCl Laemmli Tris/glycine/SDS
Criterion Stain Free
Criterion TGX
Criterion TGX Stain-Free
Criterion™ XT Bis-Tris XT XT MOPS or XT MES
Criterion XT Tris-acetate XT XT Tricine
Native PAGE Criterion Tris-HCl Native Tris/glycine
Criterion Stain Free
Criterion TGX
Criterion TGX Stain-Free
Criterion XT Tris-acetate
Peptide analysis Criterion Tris-Tricine Tricine Tris/Tricine/SDS
Isoelectric focusing (IEF) Criterion IEF IEF Anode and cathode buffers
Protease detection Criterion zymogram Zymogram Tris/glycine/SDS
dsDNA separation Criterion TBE Nucleic acid Tris/boric acid/EDTA (TBE)
ssDNA and RNA separation Criterion TBE-urea TBE-urea TBE
™
1.3 Comb Configurations
Comb Type Well Volume
12+2 we ll1 45 μl with two 15 μl reference wells
18-well 30 μl
26-well1 15 μl
Prep+2 well 800 μl with two 15 μl reference wells
IPG+1 well 11 cm ReadyStrip™ IPG strip (450 μl) with one 15 μl reference well
1.4 Specifications
Gel material Polyacrylamide
Gel dimensions (W x L) 13.3 x 8.7 cm
Gel thickness 1.0 mm
Resolving gel height 6.5 cm
Cassette dimensions (W x L) 15.0 x 10.6 cm
Cassette material Styrene copolymer
Comb material Polycarbonate
Running buffer 460 ml (per gel)
1
Multichannel pipet compatible.
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Instruction Manual and Application Guide
1.5 Storage Conditions
Table 1.2. Storage conditions for Criterion precast gels. Store gels flat. Shelf life is from date of manufacture; expiration dates
are printed on the cassettes.
Storage Temperature Gel Formulation Shelf Life
Ambient Criterion XT Bis-Tris 12 months
2–8°C Criterion TGX 12 months
Criterion TGX Stain-Free 12 months
Criterion Tris-HCl 12 weeks
Criterion Stain-Free 12 weeks
Criterion XT Tris-acetate 8 months
Criterion Tris-Tricine 12 weeks
Criterion IEF 24 weeks
Criterion zymogram 8 weeks
Criterion TBE 12 weeks
Criterion TBE-urea 8 weeks
1.6 Important Notes
Use each Criterion precast gel as soon as possible after removing it from the storage pouch.
Improper storage of Criterion precast gels can produce numerous artifacts.
n
Store gels flat
n
Avoid prolonged storage at temperatures above those recommended
n
Do not freeze gels
n
If you suspect your gels have been stored improperly, discard them
Do not run more than one gel type in the same apparatus at the same time. Different gel percentages
and formulations have different conductivities and different run times.
Use unstained standards with Criterion TGX Stain-Free and Criterion Stain Free gels, as some
™
prestained standards are not detected by the Gel Doc
EZ imager. To monitor electrophoresis, use
10 µl of a 1:1 mixture of Precision Plus Protein™ unstained and prestained standards.
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Setup and Basic
2
Operation
2.1 Workflow Overview
Prepare sample and running buffers
Assemble Electrophoresis Cell
Prepare and Load Samples
Prepare Buffers
Prepare Gels and
Dilute in sample buffer
Perform Electrophoresis
SDS-PAGE (Chapter 3)
Native PAGE (Chapter 4)
Peptide Analysis (Chapter 6)
Isoelectric Focusing (Chapter 7)
Protease Analysis (Chapter 8)
Nondenaturing Nucleic Acid PAGE (Chapter 9)
Denaturing Nucleic Acid PAGE (Chapter 10)
2-D Electrophoresis (Chapter 11)
Analyze the Separation
Blot the Gels (Optional)
(Chapter 12)
(Chapter 13)
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Instruction Manual and Application Guide
2.2 Required Materials
n
Criterion™ precast gels
n
Criterion or Criterion™ Dodeca™cell
n
PowerPac™ Basic or PowerPac HC power supply (or equivalent)
n
Sample buffer
n
Running buffer (460 ml per gel)
2.3 Setting Up and Running Criterion Gels in the Criterion Cell
1. Each Criterion gel is packaged in a plastic storage tray. Remove the cover of the tray by lifting the
corner tab and pulling it diagonally across the package. Remove the gel from the package.
2. Remove the comb and rinse the wells with deionized water (diH2O) or running buffer.
3. Remove the tape from the bottom of the cassette by pulling the tab across the gel.
4. Insert the cassette into one of the slots in the Criterion cell tank so that the integrated upper buffer
chamber faces the center of the cell (A).
5. Fill each integrated upper buffer chamber with 60 ml
running buffer.
6. Fill each half of the lower buffer tank with 400 ml
running buffer to the marked fill line.
A
Integrated upper buffer
chamber
7. Load samples using a syringe or a pipet with
gel-loading tips.
Optional: place a sample loading guide on the
outer edge of the cassette to help align pipet tips
with the wells (this is particularly useful when using
multichannel pipets).
8. Place the lid on the tank, aligning the color-coded
banana plugs with corresponding jacks on the lid.
See Chapters 3–10 for power supply settings.
2.4 Removing the Gel
1. After electrophoresis is complete, turn off the power
supply and disconnect the electrical leads.
2. Remove the lid from the tank and remove the
gel(s) from the cell. Pour off and discard the upper
running buffer.
3. Invert the cassette and place the integral buffer
chamber over the cassette-opening tool built into
the Criterion cell lid (B).
B
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Criterion Precast Gels
4. Press down firmly to break the seals on both sides of
the cassette. The cassette splits open approximately ¹/3
of the way. Alternatively, open the gel cassette by sliding
the tapered back of the comb into the slits on either
side of the cassette.
5. Pull the two halves of the cassette apart from the top to
completely expose the gel (C).
6. Remove the gel by either floating the gel into a fixing or
staining solution or by carefully lifting the bottom edge
of the gel from the cassette.
C
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3
SDS-PAGE
3.1 Introduction
Criterion™ precast gels provide versatile systems for the separation of proteins by either molecular
weight (SDS-PAGE) or mass-to-charge ratio (native PAGE). (See Chapter 4 for native PAGE applications
and protocols.) This versatility is possible because Criterion gels are made without SDS, allowing the
sample buffer and running buffer to determine the separation mechanism.
SDS-PAGE relies on a discontinuous buffer system. Two ions differing in electrophoretic mobility form
a moving boundary when voltage is applied. Proteins have an intermediate mobility that causes them
to concentrate, or stack, into a narrow zone at the beginning of electrophoresis. As that zone moves
through the gel, the sieving effect of the polyacrylamide gel matrix causes proteins of different molecular
weights to move at different rates. This stacking effect is responsible for the high resolving power of
SDS-PAGE: the sample is loaded in a relatively broad zone, and the moving boundary concentrates the
proteins into sharp bands prior to separation.
Protein samples for SDS-PAGE are prepared using SDS and usually a thiol reducing agent such as
β-mercaptoethanol or dithiothreitol (DTT). SDS forms complexes with proteins, giving them a rodlike
shape and similar mass-to-charge ratio. The reducing agent disrupts disulfide bonds between and
within proteins, allowing complete denaturation and dissociation. Heat treatment in the presence of
SDS and reducing agent effectively eliminates the effects of native charge and higher order structure on
electrophoretic mobility, so the migration distance depends primarily on molecular weight.
Molecular weight is estimated by plotting the logarithm of protein molecular weight vs. the relative
mobility (R
or by using the point-to-point semilog interpolation method in Quantity One® or Image Lab™ software.
Refer to bulletins 3133 and 3144 for more information.
) of the protein (Rf = distance migrated by the protein/distance migrated by the dye front)
f
3.2 Criterion Gel Selection Guide for SDS-PAGE
A number of Criterion gel types are available for SDS-PAGE (Table 3.1) in both single and gradient
polyacrylamide percentages. Use the protein migration charts and tables to select the gel type that
offers optimum resolution of your sample:
n
Use single-percentage gels to separate bands of similar molecular weight. Optimum
separation occurs in the lower half of the gel, so use a percentage in which the protein
migrates to the lower half of the gel
n
Use gradient gels to separate samples containing a broad range of molecular weights.
Gradient gels allow resolution of both high and low molecular weight bands on the same
gel. Larger pore sizes at the top of the gel permit resolution of larger molecules, and smaller
pore sizes toward the bottom of the gel restrict excessive separation of small molecules
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Criterion Precast Gels
Table 3.1. Criterion precast gels for SDS-PAGE.
Gel Formulation Gels Description
TGX (Laemmli-like) Criterion™ TGX™ Laemmli-like, extended shelf life gels
Criterion™ TGX Stain-Free™ Laemmli-like, extended shelf life gels with trihalo compounds for rapid
fluorescence detection
Tris-HCl (Laemmli) Criterion Tris-HCl Tris-HCl Laemmli gels
Criterion Stain Free™ Tris-HCl Laemmli gels with trihalo compounds for rapid fluorescence
detection
Bis-Tris Criterion™ XT Bis-Tris Based on a Bis-Tris HCl buffer system (pH 6.4); use these gels with
Criterion XT MES buffer for optimum resolution of small proteins
Bis-Tris Criterion XT Bis-Tris Based on a Bis-Tris HCl buffer system (pH 6.4); use these gels with
Criterion XT MOPS buffer for optimum resolution of midsized proteins
Tris-acetate Criterion XT Tris-acetate Based on a Tris-acetate buffer system (pH 7.0)
3.2.1 Criterion TGX and Criterion TGX Stain-Free Gels
Criterion TGX (Tris-Glycine eXtended shelf life) gels are Laemmli-like gels with a proprietary modification
that extends their shelf life to 12 months and enhances separation characteristics relative to conventional
gel types. The TGX formulation yields Laemmli-like separation patterns with short run times and
exceptionally straight lanes and sharp bands. TGX gels offer excellent staining quality, greater transfer
efficiency, and molecular weight estimation without the need for special, expensive buffers.
These gels are run using standard Laemmli sample buffer and Tris/glycine/SDS running buffer. Two
types of TGX formulations are available:
n
Criterion TGX — Laemmli-like, extended shelf life gels
n
Criterion TGX Stain-Free — Laemmli-like, extended shelf life gels with trihalo compounds
that allow rapid fluorescent detection of proteins with the Criterion Stain Free system,
eliminating staining and destaining steps for faster results (see Chapter 5 for more details)
Gel Composition
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Shelf life ~12 months at 2–8°C; expiration date is printed on each cassette
Gel Percentage Optimum Separation Range
7.5% 40–200 kD
10% 30–150 kD
12% 20 –120 kD
18% 10–50 kD
4–15% 20–250 kD
4–20% 10–200 kD
8–16% 20 –120 kD
10–20% 10–100 kD
Any kD™ 1 10–200 kD
1
Any kD is a unique single-percentage formulation that provides a broad separation range and short running time.
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Instruction Manual and Application Guide
3.2.2 Criterion Tris-HCl and Criterion Stain Free Gels
These Tris-HCl, Laemmli gels use discontinuous glycinate and chloride ion fronts to form moving
boundaries to stack and then separate denatured proteins by size. They are run using standard Laemmli
sample buffer and Tris/glycine/SDS running buffer.
n
Criterion Tris-HCl — Tris-HCl formulation that offer a shelf life of 12 weeks
n
Criterion Stain Free — Tris-HCl gels with unique trihalo compounds that allow rapid
fluorescent detection of proteins with the Criterion Stain Free system, eliminating staining
and destaining steps for faster results (see Chapter 5 for more details)
Gel Composition
Gel buffer 0.375 M Tris-HCl, pH 8.6
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Storage buffer 0.375 M Tris-HCl, pH 8.6
Shelf life ~12 weeks at 2–8°C; expiration date is printed on each cassette
Gel Percentage Optimum Separation Range
5 % 100–250 kD
7.5% 40–200 kD
10% 30–150 kD
12.5% 20 –12 0 kD
15% 10–100 kD
18% 10–50 kD
4–15% 20–250 kD
4–20% 10–200 kD
8–16% 20–120 k D
10–20% 10–100 kD
10.5–14% 25–200 kD
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Criterion Precast Gels
Migration charts for protein standards on Criterion Tris-HCl, Criterion TGX, and TGX Stain-Free gels.
3.2.3 Criterion XT Bis-Tris Gels
Criterion XT Bis-Tris gels are based on a Bis-Tris HCl buffer system (pH 6.4) that uses discontinuous
chloride and MES or MOPS ion fronts to form moving boundaries that stack and separate denatured
proteins by size. This chemistry of XT Bis-Tris gels allows maximum stability and consistent results with
a shelf life of at least 12 months.
Running XT Bis-Tris gels with either XT MES or XT MOPS denaturing running buffer produces different
migration patterns. A combination of these two running buffers and three XT Bis-Tris gels can generate
up to six different migration patterns for small to midsize proteins.
Gel Composition
Gel buffer Bis-Tris HCl, pH 6.4
Crosslinker 5% C
Stacking gel 4% T, 5% C
Storage buffer Bis-Tris HCl, pH 6.4
Shelf life 12 months at ambient temperature; expiration date is printed on each cassette
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Gel Percentage Optimum Separation Range
XT MES Buffer XT MOPS Buffer
10% 2.5–200 kD 14–220 kD
12% 1–30 kD 6–66 kD
4–12% 2.5–200 kD 10–300 kD
Migration charts for protein standards on Criterion XT Bis-Tris gels.
3.2.4 Criterion XT Tris-Acetate Gels
Criterion XT Tris-acetate gels are based on a Tris-acetate buffer system (pH 7.0). It uses discontinuous
acetate and Tricine ion fronts to form moving boundaries that stack and separate large denatured
proteins by molecular weight.
Gel Composition
Gel buffer Tris-acetate, pH 7.0
Crosslinker 3.8% C
Stacking gel 4% T, 3.8% C
Storage buffer Tris-acetate, pH 7.0
Shelf life 8 months at 2–8°C; expiration
date is printed on each cassette
Gel Percentage Optimum Separation range
7% 36–200 kD
3–8% 40–400 kD
Migration charts for protein standards
on Criterion XT Tris-Acetate gels.
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Criterion Precast Gels
3.3 SDS-PAGE Buffers
Table 3.2. Recommended Criterion precast gels and buffers for SDS-PAGE.
Gel Type Sample Buffer Running Buffer
Criterion TGX Laemmli (catalog #161-0737) Tris/glycine/SDS
Criterion TGX Stain-Free Optional: 2-mercaptoethanol (catalog #161-0732)
Criterion Tris-HCl (catalog #161-0710) or DTT
Criterion Stain Free (catalog #161-0611)
Criterion XT Bis-Tris XT sample buffer (catalog #161-0791) XT MES (catalog #161-0789)
Optional: XT reducing agent XT MOPS (catalog #161-0788)
(catalog #161-0792)
Criterion XT Tris-acetate XT Tricine (catalog #161-0790)
3.3.1 Running Buffers
See Appendix B for buffer formulations. Do not adjust pH.
Tris/glycine/SDS (1x) 25 mM Tris, 192 mM glycine, 0.1% SDS
(pH 8.3) Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml diH2O
XT MES (pH 6.4) Dilute 50 ml 20x stock (catalog #161-0789) with 950 ml diH2O
XT MOPS (pH 6.9) Dilute 50 ml 20x stock (catalog #161-0788) with 950 ml diH2O
XT Tricine (pH 8.2) Dilute 50 ml 20x stock (catalog #161-0790) with 950 ml diH2O
3.3.2 Sample Buffers
Laemmli 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% (catalog #161-0737)
bromophenol blue, 5% β-mercaptoethanol or 100 mM DTT (added fresh)
XT Use XT sample buffer (catalog #161-0791) and XT reducing agent
(catalog #161-0792)
3.4 Sample Preparation
1. Determine the appropriate concentration of sample to load (depends on the load volume and the
detection method used; see Chapter 12 for approximate stain sensitivities).
2. Dilute the sample with at least an equivalent volume of sample buffer with added reducing agent.
For nonreducing conditions, omit the reducing agent.
TGX and Tris-HCl Gels XT Gels (Bis-Tris and Tris-Acetate)
4.75 μl Laemmli sample buffer 5 μl XT sample buffer
0.25 μl β-mercaptoethanol 1 μl XT reducing agent
5 μl sample 1–14 μl sample
10 μl total volume Make up to 20 μl total volume with diH2O
3. Heat the diluted sample at 90–95°C for 5 min, or at 70°C for 10 min.
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