Generating Recombinant SIN viruses
1) Preparing the plasmid template for in vitro transcription.
Most SIN plasmids are linearized with Xhol restriction endonuclease for run-off transcription. Xhol is a relatively cheap enzyme so you can restrict 50-100ug of the purified SIN plasmid. Make sure the plasmid is completely digested before continuing. Following the restriction digest the plasmid is treated as follows:
1) Phenol/ chloroform extract.
2) Chloroform extract
3) Ethanol precipitate
4) Proteinase K digestion
a) Add 0.1 volumes of 10X proteinase K buffer
10X buffer = 0.5M NaCl/ 0.05M EDTA/ 0.1M Tris. Cl (pH8)
b) Add 0.1 volumes of 5% SDS
c) Add self- digested proteinase K to a final concentration of 100ug/ml
d) Incubate 37C for 1h
5) Repeat steps 1-3
6) Redissolve the DNA at a concentration of 1ug/ul in RNAse-free water.
2) In vitro transcription to generate recombinant SIN RNA genome.
In vitro transcription is an expensive undertaking. Reagents are expensive, particularly the SP6 polymerase and the RNA cap analog. Total reaction volumes should be no more than 50ul. The reactions should be set up as follows:
1.0-2.0ug DNA template (1ug/ml)
1X in vitro transcription buffer (optimized for SP6 polymerase; Promega)
1mM rATP (10mM; Promega)
1mM rUTP (10mM; Promega)
1mM rCTP (10mM; Promega)
0.5mM rGTP (10mM; Promega)
1.0uCi 3H-UTP or alpha- 32P UTP
1mM RNA cap analog 7mG5’ppp5’G (40mM: Ambion or NEB)
5mM dithiothreitol (DTT; Promega or Stratagene)
100ug/ml RNAse-free BSA (Promega)
1 unit/ul RNAsin (40u/ul;Promega)
20 units/ug of template DNA SP6 polymerase (15u/ul)
Incubate for 1hr at 39C; can use program TRCP-IV on the thermocycler. Quantitate the amount of transcript generated using the procedure outlined in Molecular Cloning (2nf ed. Sambrook, Fritsch, and Maniatis) vol. 3 E18-E19. This is important to know for transfection. It should be possible to generate 25-50 ug of RNA/ug DNA template although this is never guaranteed. The RNA should be sized to check the integrity of the RNA. RNA should be aliquoted and stored at -70C.