314
DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER
ln vitro cRNAsynthesis,
including capping
1,000 colonies, which are plated onto 9 cm Luria Broth plates ( containing
ampicillin 100 J..lg/ml).
2. When the colonies are visible, two replicas are made onto nitrocellulose
or nylon filters with both replica filters being placed on top of LB plates
(colonies side up) for about 6 hours until colonies arevisible (1 mm diameter).
3. One fllter is placed on top of an agar/glycerol plate, with the colonies side
up and kept as a master plate at -80°C (sealed with parafilm). The second
replica is submerged into 10ml Luria Broth containing ampicillin (100
J..lg/ml) and grown overnight at 37°C with shaking (300 rpm).
4. The cells are pelleted by centrifugation for 1 min and plasmids are isolated using a general plasmid (alkaline lysis) method. About 1-2 J..lg of
plasmid are digested with an appropriate restriction enzyme cutting
at the 3' end of the insert (in our case Noti). An aliquot is run through
an agarose gel to check if the digestionwas complete and the rest is extracted twice with a mixture of 0.1 M Tris pH 8 saturated phenol: chloroform: isoamylalcohol (25:24:1) and then precipitated with 0.1 volume 3M
sodium acetate and 2.5 volume cold 100% ethanol at -20°C for 2 hours.
Plasmids are then dissolved in 10 J..ll ofwater and used for in vitro transcription, using the appropriate RNA polymerase (in our case T7).
5. For in vitro transcription, the whole 10 J..ll (approx. 1 J..lg) of linearized
plasmid DNA is used and is added to 40 J..ll of the transcription mixture.
The reaction is incubated at 37°C for 1 hour.
6. Then 50 units of RN Aaseinhibitor and 10 units of DNAase I, RN Aase
free, are added to the samples and incubated for 15 minutes at 37°C
to digest the remaining DNA.
7. The samples are then extracted twice with phenol:chloroform:isoamylalcohol (25:24:1) and precipitated by adding one volume of ammonium
acetate 5 M and 2.5 volumes of ethanol, in order to remove free nucleotides.
Alternatively, after the extractions with phenol: chloroform:isoamyl alcohol, samples can be passed through RNase-free Sephadex G-50 Quick
Spin columns to remove free nucleotides, then precipitated with sodium
acetate and ethanol as described above.
8. The samples are precipitated at -80°C for at least 3 hours and then are
centrifuged for 45 min at 4°C, pellets rinsed with 70% ethanol and vacuum dried for 5 min.
DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER
ln vitro cRNAsynthesis,
including capping
1,000 colonies, which are plated onto 9 cm Luria Broth plates ( containing
ampicillin 100 J..lg/ml).
2. When the colonies are visible, two replicas are made onto nitrocellulose
or nylon filters with both replica filters being placed on top of LB plates
(colonies side up) for about 6 hours until colonies arevisible (1 mm diameter).
3. One fllter is placed on top of an agar/glycerol plate, with the colonies side
up and kept as a master plate at -80°C (sealed with parafilm). The second
replica is submerged into 10ml Luria Broth containing ampicillin (100
J..lg/ml) and grown overnight at 37°C with shaking (300 rpm).
4. The cells are pelleted by centrifugation for 1 min and plasmids are isolated using a general plasmid (alkaline lysis) method. About 1-2 J..lg of
plasmid are digested with an appropriate restriction enzyme cutting
at the 3' end of the insert (in our case Noti). An aliquot is run through
an agarose gel to check if the digestionwas complete and the rest is extracted twice with a mixture of 0.1 M Tris pH 8 saturated phenol: chloroform: isoamylalcohol (25:24:1) and then precipitated with 0.1 volume 3M
sodium acetate and 2.5 volume cold 100% ethanol at -20°C for 2 hours.
Plasmids are then dissolved in 10 J..ll ofwater and used for in vitro transcription, using the appropriate RNA polymerase (in our case T7).
5. For in vitro transcription, the whole 10 J..ll (approx. 1 J..lg) of linearized
plasmid DNA is used and is added to 40 J..ll of the transcription mixture.
The reaction is incubated at 37°C for 1 hour.
6. Then 50 units of RN Aaseinhibitor and 10 units of DNAase I, RN Aase
free, are added to the samples and incubated for 15 minutes at 37°C
to digest the remaining DNA.
7. The samples are then extracted twice with phenol:chloroform:isoamylalcohol (25:24:1) and precipitated by adding one volume of ammonium
acetate 5 M and 2.5 volumes of ethanol, in order to remove free nucleotides.
Alternatively, after the extractions with phenol: chloroform:isoamyl alcohol, samples can be passed through RNase-free Sephadex G-50 Quick
Spin columns to remove free nucleotides, then precipitated with sodium
acetate and ethanol as described above.
8. The samples are precipitated at -80°C for at least 3 hours and then are
centrifuged for 45 min at 4°C, pellets rinsed with 70% ethanol and vacuum dried for 5 min.