608113AM

September 2012

Instructions for Use

Storage of all Fragment Analysis reagents (Size
Standards, Test Sample, and Mobility Calibration
Standard) must be in a -20C non frost-free freezer.

GenomeLab
Fragment Analysis Protocol
1.

Reserve for bar code label

Preparation of the Fragments for Analysis

Prepare the PCR reactions in a thin wall microplate or tube. All
reagents should be kept on ice while preparing the PCR
reactions. Follow the recommendations of the enzyme manufacturer
for the set up of the PCR reactions.
Final concentration of the labeled primers in the PCR reaction should
be 0.25 M.

Note: Certain primers may work better at higher or lower concentrations because of
primer sequence specific factors. It is recommended to start at 0.025 M for the
System, and adjust as required.

2.

Materials Required

Thermal Cycling Program

The following touchdown procedure works well for most primer sets:
16 cycles

Materials provided by Beckman Coulter:

DNA Size Standard Kit - 400 (PN 608098)
OR
DNA Size Standard Kit - 600 (PN 608095)

94C
60C
72C

Fragment Analysis Test Sample (PN 608105)

Separation Gel I 10 mL for 2000XL/8000 Series (PN 608010)
Separation Gel I 20 mL for 8800 Series (PN 391438)
Separation Buffer (PN 608012)
DNA Separation Capillary Array, 33-75B (PN 608087)
Samples Loading Solution, SLS (PN 608082)

20 mg/ml Glycogen (Boehringer Mannheim, Cat # 901 393)

3M Sodium Acetate pH 5.2 (Sigma, Cat # S7899)
Molecular Biology Grade Sterile dH2O
Absolute 200 proof ethanol
Molecular Biology Grade 95% (v/v) ethanol / dH2O
Molecular Biology Grade 70% (v/v) ethanol / dH2O
Thermal cycler with heated lid
PCR enzyme and buffer
WellRED Dyes - Labeled primers
Thermal cycling plates and caps

45 seconds
45 seconds
(decrease by 0.5C/cycle)
30 seconds

Followed by 16 cycles:
94C
52C
72C

Required materials not supplied by Beckman Coulter:

(first annealing temperature: 60C;

last annealing temperature: 52C)

45 seconds
45 seconds
30 seconds

Note: Other primer sets may require different conditions. The presence of the
fluorescent label on one of the primers does not impact cycling conditions, therefore,
conditions used for unlabeled primers should be used for the equivalent labeled
primer sets.
Certain enzymes require an activation step. Follow the manufacturers
recommendations for activating these enzymes.

3.

Sample Preparation for Loading into the instrument:

(non-multiplexed samples)
Component
SLS
Size Standard
Sample
Total Volume

a)

Page 1 of 2

1 Sample
39.5 -X L
0.5 L
X L
~40.0 L

8 Samples
316 - X L
4.0 L
X L
~320.0 L

Add 0.25 - 3.0 L of PCR product to the SLS-size standard mix.

For optimal analysis results do not saturate the detection
system by overloading the sample separation with labeled PCR
products.
i)
For more consistent pipetting, the samples should be
diluted in SLS and loaded in a larger volume. For example;
for 0.25 L of the original sample, load 2.5 L of a 1:10
dilution.
ii) The relative signal strengths of the amplification products
vary depending on the dye label. As a guide, roughly equal
signal strengths can be obtained by adding the following
relative volume of PCR reaction products:
0.1 L D4 PCR reaction
0.2 L D3 PCR reaction
0.4 L D2 PCR reaction
Dilute the reactions appropriately to increase pipetting
accuracy. If large volumes of diluted reactions are used,
compensate to reach a final injection volume of
approximately 40 L.
iii) The amount of PCR product used will depend upon the
efficiency of the PCR reaction. The volumes given above
are a recommended starting point. Certain samples may

require that more or less volume of the PCR reaction be

added. Adding more of the PCR reaction can reduce the
signal strength of the size standard peaks and any other
sample that is co-injected with it. The effect is exaggerated
by salt in the unpurified PCR reactions, as well as the
presence of large amounts of unincorporated primers,
because all negatively charged molecules compete for
electrokinetic injection. Optimal primer incorporation and
salt removal may be required for some experiments.
4.

Sample Separation on the Instrument:

a) Several different separation methods and analysis parameter
sets are provided with the System. The Frag-1 and Frag-3
separation methods are appropriate for use with the Size
Standard 400 ladder. The Frag-2 and Frag-4 separation
methods are optimized for use with the Size Standard 600
ladder. If separations are performed with the 400 Size Standard
the Default Fragment Analysis Paramters are appropriate to
begin with. If the 600 size standard is used the default analysis
parameters will need to be modified to use the 600 size
standard set and Quartic model. Changes made to these
separation methods and analysis parameters must be optimized
by the user.
b) At 35C, the 140 nt fragment peak falls about 1 nt to the right of
a calibration plot (migration time vs. size), and should not be
included in estimating calibration curve parameters. The
anomalous mobility of this fragment is reduced at higher
separation temperatures, i.e., 50C.
c) For proper sizing of PCR products, the correct dye mobility
calibration must be chosen. Use AE-Ver2 Dye mobility
calibration for primers labeled with active ester-linked dyes and
PA-Ver1 with primers labeled with phosphoramidite linked dyes.

Fragment Analysis
Test Sample
The Fragment Analysis Test Sample is designed to test the
resolution of the instrument, as well as display peaks in all dye
colors. The Test Sample contains fragments of the following sizes:
Fragment Size
(nucleotides)

Default Dye
Color

109

D4 (blue)

110

D4 (blue)

389

D3 (green)

390

D3 (green)

589

D2 (black)

591

D2 (black)

2.

Expected Results
Verify that the six peaks of the Fragment Analysis Test Sample have
been identified and their sizes estimated, as listed in the table above.

Appendix
Appendix A
Running Pooled Samples
When running pooled samples on the instrument, it may be
necessary to adjust the amount of sample and Size Standard used.
The amount of size standard may need to be increased. This is
because the more fragments present in a sample, the greater the
competition for injection. With pooled samples, there will be more
fragments and more salt (from the PCR reaction buffer) competing
for injection into the capillary.
The amount of sample should also be increased to help overcome
the competition effect.
The simplest way to determine the amount of sample required is to
run the individual PCR reactions individually and then adjust the
amounts in the pooled samples to give balanced signals. If an
individual sample gives a high signal then the amount loaded can be
reduced more than a sample that gives low signal. Keep in mind that
all signals will be lower in the pooled sample compared to the
individual samples.
By desalting the PCR reactions prior to loading onto the instrument,
the salt competition can be reduced. Recommended desalting
procedures are given in Appendix B.

Appendix B
Desalting of PCR Reactions by Ethanol precipitation
Add 0.25 L 20 mg/mL Glycogen (Boehringer Mannheim, Cat #
901 393) to the PCR reactions and mix well.
Add 1/10 volume 3M NaOAc pH 5.2 and mix.
Add 2.5 volumes 95% (v/v) ethanol / dH2O from -20C freezer and
mix well.
Centrifuge in a microfuge at 4C for 15 minutes.
Rinse the pellet 2 times with 200 L 70% (v/v) ethanol / dH 2O from
-20C freezer. For each rinse, centrifuge immediately at 4C for a
minimum of 2 minutes.
Vacuum dry for 15 minutes.
Resuspend the samples in SLS using the same volume as the
original PCR reaction.
Note: It is recommended that purchased ready-made 3M Sodium acetate pH 5.2
solution is used, as inconsistencies in lab prepared solutions can result in
inconsistent performance on the System. Additionally, only absolute 200 proof
ethanol should be used for preparing the 95% and 70% ethanol solutions rather than
less pure or denatured sources.

Appendix C

In order to test the operation of the instrument, the following

procedure should be carried out:
1.

Preparing and Running the Fragment Analysis Test Sample

a) Prepare test samples enough for 8 wells by combining the
following in an eppendorf tube; 200 L of SLS, 8L of test
sample and 1 L of Size Standard-600. Mix the contents by
pipetting up and down a few times.
b) Fill each well of one row of the sample plate with 25 L of the
prepared test sample/size standards mix.
c) Add one drop of mineral oil to each of the 8 wells and load onto
the instrument.
d) Run the prepared sample on the instrument using the
FragTest default run method.
e) The AE-Ver1 dye mobility calibration in the analysis parameters
must be used for correct sizing of the Fragment Analysis Test
Sample.

Page 2 of 2

Size Standard Fragment Sizes

The Size Standard-400 contains fragments of the following sizes,
labeled with D1(red) dye: 60, 70, 80, 90, 100, 120, 140, 160, 180,
190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, and 420
nucleotides.
The Size Standard-600 contains fragments of the following sizes,
labeled with D1 (red) dye: 60, 70, 80, 90, 100, 120, 140, 160, 180,
190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420,
440, 460, 480, 500, 520, 540, 560, 580, 600, 620, and 640
nucleotides.

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