Eberwine T7 RNA Amplification
30 May 2002
Reagents:
Oligo-dT/T7 (Eberwine) | 5�-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T15-3� |
or T7-oligodT (Luo) | 5�-TCT AGT CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GCGT21-3� |
SuperScript II | GibcoBRL, 200 U/µl, 18064-022 |
5X First Strand Buffer | GibcoBRL, included with enzyme |
DTT | GibcoBRL, 0.1 M, included with enzyme |
Nucleotides | Pharmacia, 100 mM each of dGTP, dATP, dTTP, dCTP, 27-2035-02* |
RNase Inhibitor | Boehringer Mannheim, 40 U/µl, 799017 |
or RNasin | GibcoBRL, 40 U/µl15518-012 |
Linear Acrylamide | Ambion, 0.1 µg/µl, 9520 |
5x Second Strand Buffer | 100 mM Tris pH 6.9, 450 mM KCl, 23 mM MgCl2, 750 µM β-Nicotine Adenine Dinucleotide (Sigma, N6522), 50 mM (NH4)2SO4 |
E. coli DNA polymerase I | NEB, 10 U/µl, 209L or GibcoBRL, 10 U/µl, 18010-017 |
E. coli RNase H | GibcoBRL, 2 U/µl, 18021-071 |
E. coli DNA ligase | NEB, 10 U/µl, 205L or GibcoBRL, 10 U/µl, 18052-019 |
Phenol:Chloroform:IAA | Ambion, 25:24:1, pH 6.6, raised to pH 7.9 with included buffer (400 ml), 9732 |
Ammonium Acetate | Ambion, 5M (0.2 µm), 9070G |
Ethanol | Stores, 100% (-20°C) |
T7 MegaScript Kit | Ambion, 1334 |
or Ampliscribe T7 Kit | Epicentre, AS2607 (25x)/AS3107 (50x) |
MicroBioSpin column | BioRad, P-6 732-6221, P-30 732-6223 |
Phase-Lock Gel Light | Brinkman Instruments, 1.5 ml, 0032007.961 |
* Nucleotide mix: use 10 mM dGTP/dATP/dTTP/dCTP.
** **
Protocol:
Mix together:
200 ng poly A+ RNA (or 4-10 µg total RNA)
1 µl oligo-dT/T7 (1 µg/µl)
10 µl total volume
Heat to 70°C for 10 minutes.
Chill on ice for 30 seconds.
Centrifuge briefly.
Add the following (on ice):
4 µl 5x First Strand Buffer
2 µl DTT (0.1 M)
1 µl dNTP mix (10 mM)
1 µl Linear Acrylamide (0.1 µg/µl)
1 µl RNase Inhibitor (40 U/µl)
Note: when doing multiple reactions, make up premix containing all and aliquot 9 µl premix to each tube.
Mix contents of tube gently. Heat to 42°C for 1 minute.
Add:
1 µl SuperScript II (200 U/µl)
20 µl total volume
Mix contents of tube gently and incubate at 42°C for 1-2 hours.
Place on ice and keep cool while adding second strand components.
Add the following:
30 µl 5x Second Strand Buffer (recipe below)
3 µl dNTP mix (10 mM)
4 µl E. coli DNA polymerase I (10 U/µl)
1 µl E. coli RNase H (2 U/µl)
1 µl E. coli DNA ligase (10 U/µl)
91 µl ddH2O
150 µl total volume
Mix contents of tube gently and incubate at 16°C for 2 hours.
Add 3 µl 2.5M NaOH and incubate at 37°C for 10 minutes.
Add 150 µl buffered Phenol:Chloroform:Isoamylalcohol (25:24:1).
Mix by pipetting, spin 15K for 5 minutes.
Transfer aqueous phase to new tube.
Precipitate with 150 µl 5 M Ammonium Acetate and 1 ml 100% Ethanol.
Keep at -20°C for at least 15 minutes.
Vortex, spin 15K for 20 minutes at RT.
Add 100 µl 100% Ethanol, spin 15K for 5 minutes.
Resuspend dry pellet in 50 µl ddH2O or 10mM Tris pH 7.5.
Pass through BioRad MicroBioSpin column (P-6 5bp, P-30 20bp). Mix column, remove cap, snap off bottom, spin 2 minutes at 1000x g, change collection tube, add sample, spin 4 minutes at 1000x g.
Dry purified probe down in speedvac to 16 µl or less.
T7 amplification kit: use all 16 µl in 40 µl reaction (double volume). Add sequentially (at RT):
16 µl template DNA
4 µl 10x T7 Reaction Buffer
12 µl NTP mix (100 mM)
4 µl DTT (100 mM)
4 µl Ampliscribe T7 enzyme solution
40 µl total volume
Incubate 42°C for 2-3 hours.
Add 110 µl H2O and 150 µl water-saturated Phenol:Chloroform:Isoamylalcohol (25:24:1).
Mix by pipetting, spin 15K for 5 minutes.
Transfer aqueous phase to new tube.
Precipitate with 150 µl 5 M Ammonium Acetate and 1 ml 100% Ethanol.
Keep at -20°C for at least 15 minutes.
Vortex, spin 15K for 20 minutes at RT.
Add 100 µl 100% Ethanol, spin 15K for 5 minutes at 4°C.
Resuspend dry pellet in 30-50 µl ddH2O.
Pass through BioRad MicroBioSpin column as in step 21.
Quantitate approximate aRNA yield by Bioanalyzer and spectrophotometer (1 µl).
Ready for CyDye probe synthesis. Label at least 5 µg of amplified RNA per reaction with 3-8 µg random hexamers. Yield ~20 µg aRNA from 200ng mRNA.
5x Second Strand Buffer Recipe:
Final Concentration Stock 10 ml
100 mM Tris pH 7.0 1.0 M 1.000 ml
450 mM KCl 2.0 M 2.250 ml
23 mM MgCl2 1.0 M 0.230 ml
50 mM (NH4)2SO4 1.0 M 0.500 ml
750 µM β-NAD 0.1 M 0.075 ml
ddH2O - 5.945ml
Making 0.1 M β-NAD: 0.25(g)/663.4(mw)/0.1(M)=3.768(ml). Add 3.768 ml ddH2O to 250 mg β-NAD.
Making 50 ml 1M (NH4)2SO4: 132.14(mw) x 0.05(ml) x 1(M)=6.607 (g). Add 50 ml ddH2O to 6.607 g (NH4)2SO4.
All the other buffers are from the Ambion Buffer Kit, 9010