Biomedical Engineering Reference
In-Depth Information
2 In a new 0.2ml microcentrifuge tube, mix 15
μ
l of PCR master mix with 4
μ
lofRT
product (from step 5 of Protocol 5.
x
)and0.5
μ
l of Taq DNA polymerase.
3 To synthesize cDNA second strands, incubate in a thermal cycler as follows:
•
94
◦
C, 15 s
•
50
◦
C, 2 min
•
72
◦
C, 2 min.
4 To PCR amplify the double-stranded DNA, incubate for 35 cycles of
y
:
•
94
◦
C, 15 s
•
60
◦
C, 30 s
•
72
◦
C, 2 min.
5 In a new 0.2ml microcentrifuge tube, combine 15
μ
l of fresh PCR master mix (prepared
as described in step 1) with 3
μ
l of product from step 4. Add 1.2
μ
lof75m
M
MgCl
2
and
0.5
μ
l of Taq DNA polymerase. Complete a second round of five cycles of amplification,
as follows:
•
94
◦
C, 15 s
•
60
◦
C, 30 s
•
72
◦
C, 2 min.
6 Check the size of the PCR product
z
,
aa
by 1.8% (w/v) agarose gel electrophoresis.
Notes
x
If the RT is performed on a single cell, or less than 20 pg of total RNA, then 4
μ
l of the RT
product are added to each of three tubes containing PCR master mix, so that nearly all of the
RT reaction is carried forward to amplification. This is done to increase the chances that genes
that are not highly expressed will be sufficiently amplified to maintain their representation
when starting with a small pool of RNA.
y
Create one PCR program to complete both the second-strand cDNA synthesis (step 3) and the
PCR amplification of double-stranded DNA (step 4).
z
The PCR product should appear as a smear between about 200 and 400 bp.
aa
Store the double-stranded PCR product at
−
20
◦
C.
PROTOCOL 5.10 Aminoallyl Incorporation
Equipment and reagents
•
Recombinant Taq DNA polymerase and included reagents (Taq DNA polymerase, 10
×
Taq
buffer, 50m
M
MgCl
2
; Life Technologies)
SR-T24 primer (5
-GTT AAC TCG AGA ATT CTT TTT TTT TTT TTT TTT TTT TTT T-3
;7
μ
M
)
•
