Biomedical Engineering Reference
In-Depth Information
9.4
Generation of PCR Fragments from Plasmid DNA
as a Template for RNA Probe Synthesis
1. Amplifi cation of PCR fragment that includes the gene of interest (GOI) and RNA
polymerase binding sites, such as T7, T3, and Sp6 sequences, at both ends of GOI.
For this purpose, we regularly use pGEM-T Easy Vector that possesses T7 and
Sp6 sites, M13For and Rev sites, and restriction enzyme cloning sites (Fig.
9.2
).
Probe size, i.e., GOI size, can be adapted from 150 to 3,000 bp.
2. Perform PCR reaction.
2.1 PCR content
2 0
μ
L of distilled water (GIBCO, cat#10977)
1
g) of DNA template, e.g., pGEM-T easy plasmid DNA includ-
ing GOI {
Note*1
}
1.5
μ
L (−0.1
μ
μ
L of M13Rev oligo primer (5
′
-ACAGGAAACAGCTATGACC-3
′
:
20
μ
M)
1.5
μ
L of M13For oligo primer (5
′
-TGTAAAACGACGGCCAGT-3
′
:
20
μ
M)
3
μ
L of 10× DNA polymerase buffer
2.5
μ
L of 2.5 mM each dNTPs (included with Taq polymerase kit)
0.5
μ
L of Taq DNA polymerase [5 unit/
μ
L, Takara Ex taq cat#RR001]
Subtotal 30
μ
L in a tube × four tubes = total 120
μ
L
Fig. 9.2
Flow chart for making RNA probe synthesis from DNA plasmid
