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is terminated with TTTTT sequence which constitutes the pol
III terminator (Fig.
1
). The antisense strand oligo is with the
exception of its
Eco
RI overhang (AATT) at the 5
′
end 100 %
complementary to the sense oligo.
4 Generation of Inserts by Amplifi cation of Genomic Regions Containing
a pri-miR Sequence
1. Amplify the pri-miR from (human) genomic DNA using prim-
ers with
Age
I and
Eco
RI adaptors. Use a proof-reading DNA
polymerase.
2. Review outcome (specifi city) of the PCR by applying an aliquot
to an agarose gel (1.5 %).
3. Purify PCR reaction from polymerase using standard phenol-
chloroform extraction and exchange buffer by standard
precipitation (
see
Subheading
4.2
).
4. Digest the PCR product with
Age
I and
Eco
RI.
5. Gel-purify the amplicon of the pri-miR.
4.1 Generation
of Inserts by
Hybridization of
Oligonucleotides
1. Dissolve the lyophilized molecules in TE to a fi nal concentra-
tion of 100
M.
2. Mix complementary oligonucleotides together at a 1:1 M ratio
in a 0.5 mL reaction tube:
-1.5
μ
μ
L sense oligo.
-1.5
μ
L antisense oligo.
-10
μ
L 5× ligase buffer.
L H
2
O (
see
Note 6
).
3. Perform the annealing reaction in a PCR-cycler using the
following protocol (
see
Note 7
):
-37
μ
99 °C
1 min
96 °C
7 min
85 °C
5 min
75 °C
7 min
65 °C
10 min
37 °C
10 min
22 °C
20 min
16 °C
10 min
6 °C
Hold
