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September 2012
GenomeLab
Fragment Analysis Protocol
1.
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
94C
60C
72C
45 seconds
45 seconds
(decrease by 0.5C/cycle)
30 seconds
Followed by 16 cycles:
94C
52C
72C
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.
a)
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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
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
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