- Oxidizer: 0.02 M iodine in pyridine, water, and THF
0.4:9:90.6 (v:v:v) (e.g., emp Biotech). - Deblock: 6% dichloroacetic acid in methylchloride (e.g., emp
Biotech). - Acetonitrile for DNA-synthesis.
2.2 Probe Cleavage
and Purification
- 32% aqueous ammonia.
- 55C rocking heating block.
- 80% aqueous AcOH.
4.iProOH. - 3Mammonium acetate.
- Reversed Phase HPLC e.g., 1105 HPLC System from Gilson,
using a Waters X-Bridge BEH130 C18 column (10150 mm,
5 μm) at a flow rate of 8 mL/min at 55C.
(DMTr-on: 15–40% B in 10 min, A¼0.1 M triethylammo-
nium acetate, aq. pH 7.4; B¼MeCN)
(DMTr-off: 5–20% B in 10 min, A¼0.1 M triethylammonium
acetate, aq. pH 7.4; B¼MeCN)
2.3 Probe Analytic 1. Reversed Phase HPLC, e.g., 1105 HPLC System from Gilson
by using a Waters X-Bridge BEH130 C18 column
(4.6250 mm, 5μm) at a flow rate of 1.5 mL/min at 55C.
(10–50% B in 10 min, A¼0.1 M triethylammonium acetate,
aq. pH 7.4; B¼MeCN).
- MALDI-TOF mass spectra (e.g., Shimadzu Axima Confi-
dence) in positive mode with HPA matrix (1:1 mixture
of¼50 mg/mL 3-hydroxypicolinic acid in MeCN/H2O 1:1
and 50 mg/mL diammonium citrate in MeCN/H2O 1:1).
2.4 In Vitro
Measurements
- Fluorescence spectrometer (e.g., Varian Cary Eclipse).
- 10 mm quartz cuvettes.
- Phosphate buffer: 10 mM Na 2 HPO 4 , 100 mM NaCl, pH 7.0.
- UV–Vis spectrometer (e.g., Varian Cary Bio 100).
- ATTO 520 and ATTO 590 fluorescent dyes.
2.5 Fly Husbandry
and Dissection
Flies are raised on standard cornmeal agar at 25C in humidified
incubators.- Dissection microscope station equipped with CO 2 pistol and
pad to anesthetize the flies. - 100% ethanol.
- Dissection buffer (BRB80): 80 mM PIPES–KOH, pH 6.9,
1 mM EGTA, and 2 mM MgCl 2.
In vivo mRNP Detection by FIT Probes 275