9. Resuspend the pellet in 10 mL diethyl ether and centrifuge at
4470 Â g for 2 min. Pour off the supernatant, and use a
lyophilizer or a vacuum oven at 40
C to dry the pellet overnight. After dry, the ImpA powder can be stored in a sealed
bottle at À80
C up to 1 week.
Fig. 2 Synthesis of the 5
0
adenylated 3
0
adapter oligonucleotides. (a) Setting up an O 2 -free reaction system
using a balloon with an excess of N 2 (in flask B). (b) Adding solution A dropwise to solution B. (c) Detection of
synthesized ImpA through TLC. The product pointed by an arrow indicates the synthesized ImpA. (d)
Visualization of adenylated adapter through UV shadowing. The adenylated adapter (upper band) and the
unadenylated adapter (lower band), and the silica gel plate are marked by black arrows. In addition to recovery
of the pre-adenylated product, the unadenylated adapter should also be recovered for a repeat assay when
needed
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Di Sun et al.
4470 Â g for 2 min. Pour off the supernatant, and use a
lyophilizer or a vacuum oven at 40
C to dry the pellet overnight. After dry, the ImpA powder can be stored in a sealed
bottle at À80
C up to 1 week.
Fig. 2 Synthesis of the 5
0
adenylated 3
0
adapter oligonucleotides. (a) Setting up an O 2 -free reaction system
using a balloon with an excess of N 2 (in flask B). (b) Adding solution A dropwise to solution B. (c) Detection of
synthesized ImpA through TLC. The product pointed by an arrow indicates the synthesized ImpA. (d)
Visualization of adenylated adapter through UV shadowing. The adenylated adapter (upper band) and the
unadenylated adapter (lower band), and the silica gel plate are marked by black arrows. In addition to recovery
of the pre-adenylated product, the unadenylated adapter should also be recovered for a repeat assay when
needed
236
Di Sun et al.