Biomedical Engineering Reference
In-Depth Information
—SALSA enzyme dilution buffer
—SALSA polymerase
Thermocycler (equipped with a heated lid)
ii
programmed to perform the complete MLPA
program:
—Hybridization program
◦
98
◦
C, 5 min
◦
25
◦
C, hold
◦
95
◦
C, 1 min
◦
60
◦
C, hold.
—Ligation program
◦
54
◦
C, hold
◦
54
◦
C, 15min
◦
98
◦
C, 5 min
◦
4
◦
C, hold.
—PCR program
•
60
◦
C, hold
◦
(95
◦
C, 30 s; 60
◦
C, 30 s; 72
◦
C, 1 min)
×
35
◦
72
◦
C, 20min
◦
4
◦
C, hold.
◦
Method
1 In a pre-PCR location, measure the concentrations of the stock DNA solutions (see
Protocol 1.3).
2Dilute
jj
,
kk
the DNA samples to working stocks of 10-20 ng/
μ
lusing10m
M
Tris-HCl, pH
8.0, 1 m
M
EDTA.
3 Label 0.2ml strip tubes with sample initials and probe set number.
4Add5
μ
l of a working stock DNA solution to each tube (or water for a water
blank).
5 Centrifuge the strip tubes briefly to collect the sample DNA at the bottom.
6 Put the tubes into the thermocycler and start the MLPA program (5 min at 98
◦
C); allow
the samples to cool to 25
◦
C before opening the thermocycler.
7 Remove the probe mix and the MLPA buffer from the
−
20
◦
C freezer and allow to thaw.
Vortex briefly.
8 Prepare a probe master mix by combining (per DNA sample) 1.5
μ
l of probe mix and
1.5
μ
l of MLPA buffer.
9 When the thermocycler reaches 25
◦
C, add 3
μ
l of probe master mix to each DNA sample.
Mix well by pipetting up and down.
