Generating recombinant alphaviruses
1) Preparing the plasmid template for in vitro transcription.
Overdigest the plasmid prep with the appropriate restriction enzyme, but avoid star activity. Make sure the plasmid is completely digested before continuing. Following the restriction digest, the plasmid is treated as follows:
1) Proteinase K digestion (Proteinase K is active in most buffers used in restriction digestion reactions.) Alternatively, linearized DNA may be cleaned and digested as described below (a-d).
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
2) Phenol/ chloroform extract
3) Chloroform extract
4) Ethanol precipitate
5) Redissolve the DNA at a minimum concentration of 1ug/7.5ul of RNAse-free water.
2) In vitro transcription to generate recombinant alphavirus RNA.
In vitro transcription is an expensive undertaking. Reagents are expensive, particularly the RNA polymerase and the RNA cap analog. Total reaction volumes should be no more than 50ul. The reactions should be set up at the following final concentrations:
1.0-2.0ug DNA template (1ug/ml)
1X in vitro transcription buffer (optimized for SP6 or T7 polymerases)
1mM rATP
1mM rUTP
1mM rCTP
1mM rGTP
1mM RNA cap analog 7mG5’ppp5’G or 7mG5’ppp5’A
5mM dithiothreitol (DTT)
100ug/ml RNAse-free BSA
1 unit/ul RNAsin
20 units/ug of template DNA SP6 or T7 polymerase
Rnase-free H20 as needed
Incubate for 1hr at 39C; can use program TRCP-IV on the thermocycler. The amount of transcript generated can be quantified using a procedure outlined in Molecular Cloning (2nf ed. Sambrook, Fritsch, and Maniatis) vol. 3 E18-E19. This may important to know depending on the experiment. It should be possible to generate 25-50 ug of RNA/ug DNA template, although, this is never guaranteed. The integrity of the RNA may be checked by sizing. RNA should be aliquoted and stored at -70C.
