Materials and Reagents

1. For in vitro transcription (IVT) in general
   1. T7 RNA polymerase including 5x T7 transcription buffer (Thermo Fisher Scientific, Fermentas, catalog number: EP0111 )
      
   2. RiboLock RNase inhibitor (Thermo Fisher Scientific, Fermentas, catalog number: EO0381 )
      
   3. 100 mM of ATP, UTP, GTP, CTP (NTP set) (Thermo Fisher Scientific, Fermentas, catalog number: R0481 )
      
   4. Recombinant DNase I (Life Technologies, Ambion^®, catalog number: AM2235 )
      
   5. RNase-free water (Life Technologies, Ambion^®, catalog number: AM9932 )
      
   6. [alpha ³²P]-CTP (800 Ci/mmol, 10 µCi/µl) (PerkinElmer, catalog number: BLU008X )
      
   7. 1x T7 transcription buffer (see Recipes)
      Note: Templates will be generated by PCR, so all reagents for PCR are needed, too.
      
      
2. For tRNA IVT in particular
   1. MinElute PCR Purification Kit (QIAGEN, catalog number: 28004 )
      
   2. Guanosine 5’-monophosphate disodium salt hydrate from yeast (GMP) (as 100 mM stock solution in RNase-free water) (Sigma-Aldrich, catalog number: G8377 )
      
   3. Bio-Spin chromatography columns, Bio-Gel P-6 in Tris buffer (Bio-Rad Laboratories, catalog number: 732-6227 )
      
   4. OptiPhase Supermix liquid scintillation cocktail (PerkinElmer, catalog number: 1200-439 )
      
   5. Microbeta Starter kit plates (PerkinElmer, catalog number: 1450-486 )
      
      
3. For denaturing Urea-PAGE
   1. Urea (Sigma-Aldrich, catalog number: U6504 )
      
   2. Rotiphorese 40% acrylamide/bis-acrylamide (19:1) solution (Carl Roth, catalog number: 3030.1 )
      
   3. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: 215589 ) [as 10% (w/v) solution in RNase-free water]
      
   4. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
      
   5. 2x MOPS buffer (e.g. Life Technologies, Ambion^®, catalog number: AM9570 )
      
   6. Formamide (e.g. Life Technologies, Ambion^®, catalog number: AM9342 )
      
   7. Formaldehyde (e.g. Sigma-Aldrich, catalog number: F8775 )
      
   8. Saccharose (e.g. Sigma-Aldrich, catalog number: S7903 )
      
   9. Bromophenol blue (e.g. Sigma-Aldrich, catalog number: B0126 )
      
   10. Xylencyanol (e.g. Sigma-Aldrich, catalog number: X4126 )
       
   11. 1x TBE buffer (see Recipes)
       
   12. 2x RNA loading dye (see Recipes)
       
   
   
4. For native polyacrylamide gel electrophoresis (PAGE)
   1. 40% acrylamide/bis-acrylamide (19:1) solution, APS and TEMED as listed in C
      
   2. Glycerol (e.g. Sigma-Aldrich, catalog number: G5516 )
      
   3. Tris (e.g. Sigma-Aldrich, catalog number: T1503 )
      
   4. Boric acid (e.g. Sigma-Aldrich, catalog number: B7901 )
      
   5. EDTA (e.g. Sigma-Aldrich, catalog number: E9884 )
      
   6. 0.5x TBE buffer (see Recipes)
      
   7. 10x loading buffer (see Recipes)
      

Equipment

1. Basic lab equipment for molecular biology with emphasis on RNase-free
   1. 0.5 ml RNase-free microfuge tubes (Applied Biosystems^®, catalog number:  AM12300 )
      
   2. 1.5 ml RNase-free microfuge tubes (Applied Biosystems^®, catalog number:  AM12400 )
      
   3. Microliter pipettes (e.g. Eppendorf) with tips and 15 ml or 50 ml tubes (e.g. SARSTEDT AG)
      
   4. Table-top centrifuge (e.g. Microcentrifuge, Eppendorf, catalog number:  5425 )
      
   5. Heating block for 1.5 ml and 0.5 ml tubes (e.g. Grant Instruments, Grant bio PCH-1 personal benchtop cooler/heaters)
      Note: Templates will be generated by PCR, so a standard PCR thermo cycler is needed, too.
      
   
   
2. Lab equipment in designated area for radioactive work
   1. Liquid scintillation counter (PerkinElmer, model: 1450 LSC & Luminescence counter)
      
   2. Electrophoresis chamber, glass slides, spacers, clamps and combs for vertical PAGE (e.g. C.B.S. Scientific, catalog number: WSP2-SG-200 ), power supply (e.g. C.B.S. Scientific, catalog number: EPS-200-X)
      
   3. Plastic wrap, transparent sheets and Whatman paper (e.g. Sigma-Aldrich, catalog number: Z742422 )
      
   4. Gel dryer (Model 583 Gel Dryer) (Bio-Rad Laboratories, catalog number: 165-1746 ) with vacuum pump
      
   5. PhosphorImager (Fujifilm FLA-7000) (GE Healthcare, catalog number: 28-9558-09 ) and PhosphorImager screen (storage phosphor screens) (VWR International, catalog number: 28-9564 ) in cassette (VWR International, catalog number: 63-0035 )
      

Procedure