Biomedical Engineering Reference
In-Depth Information
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If the stock DNA solution concentrations are less than 10 ng/
μ
l, use the available concentra-
tion, but note that results may be less reliable. 50-100 ng of DNA is recommended for each
assay, although the acceptable range is 20-500 ng of DNA.
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Both positive and negative controls (e.g. normal DNA and water respectively) can be processed
in parallel.
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All MLPA reaction mixtures should be prepared less than 1 h before use and stored on ice.
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All reagents should be returned to the freezer after usage, reducing the loss of activity of
the enzymatic solutions.
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Make master mix solutions for all reactions in an experiment in order to minimize sample
to sample variation. Add 10% more than required of each reagent to account for pipetting
loss.
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When performing MLPA on large sample numbers, multichannel pipettes are recommended.
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For longer periods, storage at
−
20
◦
C is recommended.
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PCR products can be stored at 4
◦
C for at least 1 week. As the fluorescent labels used are light
sensitive, the PCR products should be stored in a dark box, or wrapped in aluminum foil.
PROTOCOL 1.10 Separation and Relative Quantification
of MLPA Products
Equipment and reagents
•
Capillary sequencer, or slab gel DNA sequencer, with fragment analysis software; for
example
—ABI-310 (1 capillary) - capillary: 5-47 cm, 50
μ
m (ABI 402839); polymer: POP-4 (ABI
4316355) or POP-6 (ABI 4306733)
—ABI-3100 (16 capillaries), or ABI 3100 Avant (four capillaries) - capillaries: 36 cm;
polymer: POP-4 (ABI 4316355)
—ABI-3700 (96 capillaries) - capillaries: 3700 capillary array, 50 cm (ABI 4305787);
polymer: POP-4, or POP-6
•
Deionized formamide (ABI, 4311320)
•
Labelled size standard (ROX-500 ABI GeneScan 401734; TAMRA-500 ABI GeneScan
401733).
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Method
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,
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ABI-310:
1 Following the PCR reactions, for each reaction mix:
0.75
μ
l of the PCR reaction
•
0.75
μ
l of water
•
