working with RNA
treat all solutions with DEPC
store pipet tips, reaction tubes and buffers in a separate
cabinet/drawer
wear gloves, the fingers are a very good source for RNase
RNase is resitent to autoklaving! Sterile tubes may easily be
contaminated with RNase, when touched by fingers before autoklaving
Linearisation of the plasmid
template
- digest 5 to 10 µg of plasmid with an appropriate
enzyme
- increase volume with TE to 50 µl
- extract with phenol/chloroform and with chloroform
- add 0,1 vol 3 M Na-Ac, 2,5 vol cold ethanol, mix
- precipitate DNA at -20ºC
- spin 15 min, wash with cold 70% ethanol, dry pellet
- disolve in 10 µl DEPC H2O
- measure absorption at 260 nm
- store at -20ºC in RNA box
purity of plasmid: you may use minipreps
synthesis with defined components
reaction mixture (20 µl):
1 - 2 µg linearised plasmid (dep. on
size)
4 µl 5x buffer
2 µl DTT (100 mM)
1 µl RNAsin (20 u)
4 µl 5x rNTP mix (with cap)
1 µl SP6 polymerase (20-40 u)
- incubate reaction mix for 1 h at 40ºC
- add 2 µl 5mM GTP, incubate 15 min
- add 1 µl 0,5 M EDTA to stop the reaction
- at TE to a volume of 50 µl
- extract first with phenol/chloroform and then with chloroform
- add 0,1 vol 3 M Na-Ac (DEPC treated)
- 2,5 vol cold ethanol
- precipitate at -20ºC
- spin 15 min at 4ºC
- wash with 70% ethanol
- dry in speed vac
- dissolve in 10 µl DEPC H2O
- measure concentration, adjust to 1 mg/ml
- store at -20ºC (RNA box)
The typical yield is 20 µg. To remove the DNA template you may
incubate with DNAse (1U) for 15 min prior to phenol extraction.
5x buffer:
200 mM Tris/HCl pH7.5
30 mM MgCl2
10 mM Spermidin
5x rNTP mix:
2,5 mM ATP
2,5 mM UTP
2,5 mM CTP
0,5 mM GTP
2,5 mM m7G(5)ppp(5)G
RiboMAX- high
yield synthesis
The reaction mixture contains pyrophosphatase. Pyrophosphate is a
product of the polymerisation reaction and becomes rate-limiting under
standard conditions. The concentration of the nucleotides is the second rate-limiting factor.
For T7 RNA polymerase upto 7.5 mM, for SP6 upto 5 mM of each NTP can be used.
reaction mixture (20 µl)
x µl DEPC water
1-2 µg DNA template (1 µg for short PCR products)
4 µl 5x transcription buffer
x µl rNTP mix (use upto 7.5/5 mM final)
1 µl RNAsin (20 U)
1 µl PPase (0,1 U)
2 µl RNA polymerase (40 U)
- set up the reaction mixture on ice, mix
- incubate for 4h at 37°C
- add an additional 20-40 U of RNA polymerase
- incubate for 2h at 37°C
- add TE to a final volume of 100 µl
- extract with Phenol/chloroform and precipitate with ethanol
5x transcription buffer
400 mM Hepes/KOH pH 7,6
10 mM spermidine
200 mM DTT
80 mM MgCl2 for SP6 (60 mM for T3, T7)
Pyrophosphatase
dissolve 100 U of yeast PPase
(Sigma) in 100 µl of 2x PP buffer (20 mM Tris-Acetat pH 7,6, 20
mM NaCl, 0,2 mM EDTA, 2 mM DTT, 0,02% Triton X-100). Add 100 µl
of glycerol to final 50%. The final concentration of 0,5 U/µl is
100x concentrated.
The reaction conditions slightly vary for the SP6 and T3/T7 RNA
polymerase. For the SP6 pol Mg2+ is 16 mM and 1µg DNA template is
sufficient. For T3/T7 12 mM Mg and 2 µg DNA template are added.
The expected yield is in the range more than 100 µg with T7 polymerase.
Ambion kit: mRNA synthesis
x µl DEPC treated water
y µl template DNA (1 µg, linearised plasmid)
10 µl 2xNTP/cap
2 µl 10x buffer
2 µl enzyme mix
-------
20 µl
- mix, incubate at 37°C for 1 to 2 h
- add 80 µl DEPC water and 15 µl ammonium
acetate to stop the reaction
- extract with phenol/chloroform and chloroform
- precipitate with Ethanol (0.1 volume 3 M Na-Ac,
2,5 vol EtOH), mix, store overnight at -20°C
- wash with 70% ethanol
- dissolve dry pellet in 20 µl DEPC water
- measure A260 with 1 µl in 200 µl TE
- store in aliquots at -20°C
Ambion kit: RNA synthesis
x µl DEPC treated water
y µl template DNA (1 µg, linearised plasmid)
2 µl ATP (75 mM for T7 kit, 50 mM for SP6 kit)
2 µl GTP
2 µl CTP
2 µl UTP
2 µl 10x buffer
2 µl enzyme mix
-------
20 µl
- mix, incubate at 37°C for 1 to 2 h
- add 80 µl DEPC water and 15 µl ammonium
acetate to stop the reaction
- extract with phenol/chloroform and chloroform
- precipitate with Ethanol (0.1 volume 3 M Na-Ac,
2,5 vol EtOH), mix, store overnight at -20°C
- wash with 70% ethanol
- dissolve dry pellet in 50 µl DEPC water
- measure A260 with 1 µl in 200 µl TE
- store in aliquots at -20°C
template: cut the plasmid with an appropriate
enzyme, extract with phenol/choloroform and precipitate with EtOH
dissolve in DEPC water, measure A260 to determine the concentration.
Following the reaction you may add DNase to degrade the template.
yield: You may expect a yield of upto 200 µg RNA.
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