Biomedical Engineering Reference
In-Depth Information
2 Pipette this mixture into the injection plate.
3 Seal the injection plate with plate sealing film and incubate at 92
◦
C for 2min and hold
at 4
◦
Cfor5min.
4 Start the fragment separation software using the following settings:
•
sample volume: 2.5
μ
l
•
injection time: 10 s
•
injection voltage: 10 kV
•
run voltage: 7.5 kV
•
run time: 4500 s
•
cuvette temperature: 48
◦
C
•
run temperature: 50
◦
C
•
filter set: D
Notes
rr
A description of the methods and settings for a number of commercially available capillary
sequencers is provided.
ss
The amount of the MLPA PCR required for analysis by capillary electrophoresis depends on the
apparatus and fluorescent label used.
tt
Label for MLPA: SALSA 6-FAM PCR primer-dNTP mix.
PROTOCOL 1.11 Normalization of MLPA Fragment Separations
Results into Copy Number Ratios
Equipment and reagents
Capillary sequencer, or slab gel DNA sequencer, with fragment analysis software
•
The MLPA probe mix list containing the genomic map coordinates of the probes
•
The size-called fragment list(s) containing the size-called fragment lengths, heights and
areas of samples, and their references
uu
•
•
Software which calculates ratios, links the genomic position of the probes and creates
charts and reports for interpretation (e.g. Microsoft Excel, or dedicated software such as
MLPA-DAT, Genemarker or Seq Pilot)
Method
vv
1 Load the probe list of your specific MLPA mix into the program.
2 Import your fragment list and define your reference data.
ww
