Biomedical Engineering Reference
In-Depth Information
10 Continue the program on the thermocycler to advance from the 25
◦
C hold step,
incubating for 1 min at 95
◦
C, and then for 16 h (overnight) at 60
◦
C.
11 In a pre-PCR location, remove the Ligase-65 buffer A and Ligase-65 buffer B from the
freezer and allow to thaw. Vortex briefly.
12 Prepare
ll
,
mm
,
nn
,
oo
a ligase buffer mix by combining (for each reaction): 3
μ
l of Ligase-65
buffer A, 3
μ
l of Ligase-65 buffer B and 25
μ
l of water. Mix by vortexing.
13 Add 1
μ
l of Ligase-65 (per reaction) to the ligase buffer mix and mix well by
vortexing.
14 Continue the program on the thermocycler to advance to the 54
◦
C hold step.
15 When the samples are at 54
◦
C, add 32
μ
l of the ligase buffer plus Ligase-65 mix to each
reaction tube and mix well by pipetting up and down.
16 Continue the program on the thermocycler to advance to the next step (15 min
incubation at 54
◦
C, followed by 5min at 98
◦
C, and holding at 4
◦
C). Store
pp
the ligation
products at 4
◦
C for up to 1 week.
17 In a pre-PCR location, remove 10
×
SALSA PCR buffer, SALSA PCR-primers and SALSA
enzyme dilution buffer from the freezer and allow to thaw. Vortex briefly.
18 Label new tubes for PCR with the same sample initials and probe set numbers used for
the hybridization and ligation reactions.
19 Prepare
ll
,
mm
,
nn
,
oo
a PCR buffer mix by combining (per ligation product): 4
μ
lofSALSA
PCR buffer and 26
μ
l of water. Mix by vortexing.
20 Add 30
μ
l of the PCR buffer mix to each new tube.
21 Transfer 10
μ
l of each ligation product to its corresponding PCR tube.
22 Centrifuge the strip tubes briefly in order to collect the reaction mixtures at the bottom
of each tube.
23 Place tubes into the thermocycler.
24 On ice, prepare a PCR master mix combining (per ligation product): 2
μ
lofSALSA
primers, 2
μ
l of SALSA enzyme dilution buffer and 5.5
μ
l of water. Mix well by pipetting
up and down.
25 Add 0.5
μ
l of SALSA polymerase to each reaction and carefully mix again by pipetting up
and down.
26 Continue the program on the thermocycler to advance to the 60
◦
C hold step.
27 Add 10
μ
l of PCR mix to each PCR tube sitting in the thermocycler at 60
◦
C. Mix by
pipetting up and down.
28 Continue the program, proceeding to exponential amplification.
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Notes
ii
If no heated lid is available in the PCR machine, overlay each DNA sample with 15
μ
l of mineral
oil in order to prevent evaporation.
