RNA Detection

autoclaved water. Sterilize by filtering HBS solution using

sterile 0.2μm syringe filter inside the cell culture hood and

store at 4C(seeNote 1).

2. 1 Phosphate buffer saline (1 PBS): 10 mM phosphate
   buffer containing 138 mM NaCl, 2.7 mM KCl, pH 7.4. Dis-
   solve one pack of PBS powder in 1000 mL of Milli-Q water.
   Autoclave 1 PBS and then filter through sterile 0.2 μm
   syringe filter inside the cell culture hood. Aliquot 1PBS in
   50 mL sterile Falcon tubes inside biosafety cabinet and store at
   4 C(seeNote 1).


3. Tris buffered saline (TBS): 50 mM Tris base, 150 mM NaCl,
   pH 7.4. Dissolve 6.05 g of Tris base and 8.76 g of NaCl in
   1000 mL graduated cylinder containing 800 mL of autoclaved
   water. Adjust the pH to 7.4 by slowly adding 1 M HCl. Adjust
   the volume to 1000 mL by adding autoclaved water. Aliquot
   the TBS solution in 50 mL Falcons and store at 4C. Thaw
   TBS buffer at RT prior to further use.

2.2 Cell Culture 1. CO 2 cell culture incubator.

2. Cell culture dishes.


3. Sterile coverslips.


4. Dulbecco’s modified Eagle’s medium (DMEM) in 10% fetal
   bovine serum (FBS): Aliquot 45 mL of plain DMEM in sterile

Fig. 1Chemical structure of 5-azidomethyl UTP (AMUTP) and fluorescent dyes,

Alexa 594 alkyne and Cy3 DBCO

Imaging Newly Transcribed RNA in Cells 361