2. Annealing buffer: 10 mM Tris–HCl, 1 mM EDTA, 100 mM
NaCl, pH 7.8. Weigh 12.11 mg of Tris base and 58.44 mg of
NaCl in a 15 mL centrifuge tube. Add 8–9 mL of water and
20 μL of 0.5 M EDTA. Adjust the pH to 7.8 by adding 1 M
HCl. Make the final volume to 10 mL using autoclaved water
and check the pH again.
3. 20 U/μL T7 RNA polymerase stock (see Note 1).
4. NTPs minus UTP mix: 20 mM Stock solution in autoclaved
water. Mix 20 μL of 100 mM stock solutions of guanosine
triphosphate (GTP), cytidine triphosphate (CTP), and adenosine triphosphate (ATP) in a 1.5 mL microcentrifuge tube.
Make it to 100 μL by adding 40 μL autoclaved water. Vortex,
centrifuge, and store the NTP-UTP mix at À40
C.
5. 20
mM
5-Iodouridine-5
0 -triphosphate
(IUTP
1,
ε 260 ¼ 3100 M
À1 cm
À1 , can be purchased from a supplier):
Prepare this stock solution in autoclaved water. Store the stock
solution at À40
C.
6. 200 mM MgCl 2 : Weigh 40.66 mg of MgCl 2 in a 1.5 mL
microcentrifuge tube. Dissolve the solid in 1 mL of autoclaved
water. Store the solution at À40
C.
7. 40 U/μL RiboLock (see Note 1).
Fig. 2 (a) Chemical structure of boronic acid/ester substrates used in the posttranscriptional Suzuki-Miyaura
coupling reactions. (b) Ligand L1 and L2
476
Manisha B. Walunj and Seergazhi G. Srivatsan