Biology Reference
In-Depth Information
Fig. 1
Typical primer design considerations for a hypothetical four-exon gene. A good set of primers
might be the pairs indicated by a/b or c/d. The 3
0
primer carries the recognition sequence for T7 RNA
polymerase, 5
0
-TAATACGACTCACTATAGGG-3
0
, followed by 25-30 bases of target homology; the
forward primer has 25-30 bases of identity but lacks the T7 tag.
complementary (sense) strand as a control probe, or use one of the suggested probes
(Section 4) for specificity.
B. PCR to Generate Probe Synthesis Template
Assemble the following in a 0.6-mL (PCR) tube:
Forward primer (25 pmol/
m
L)
1
m
L
Reverse primer (25 pmol/
m
L)
1
m
L
dNTPs (10 mM)
0.5
m
L
PCR buffer (10
stock)
2.5
m
L
Genomic DNA (200 ng/
m
L)
a
1
m
L
Taq polymerase
b
0.5
m
L
ddH
2
O
18.5
m
L (total volume: 25
m
L)
a
Alternatively, use 1
m
L of a solution carrying 10 ng of plasmid template.
b
We routinely use a crude Taq preparation with good results (
Engelke et al., 1990
).
Perform a standard PCR reaction, for example 95
C for 3 min and then repeat
[95
C for 30 s, 72
C for 30 s, 55
C for 30 s] for 30 cycles, 72
C for 10 min and
ending at 4
C. Check an aliquot (5
m
L) on an agarose gel to make sure the PCR
product is of the expected size.
C. DIG-Labeled Probe Synthesis
Use DIG-RNA labeling kit (Roche, #1175025)
PCR product generated above (not purified)
3
m
L
10
NTP mixture with DIG-11-UTP
1
m
L
10
Transcription Buffer
1
m
L
RNase Inhibitor
0.5
m
L
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