P-MEXP-1959 - P-MEXP-1959

Type
labeling
Description
Materials 1. RNase Out, 40 U/µL (Invitrogen 10777-019) 2. HT7T-Primer, HPLC-purified, 200 ng/µL. Sequence: 5'-GGC. CAG.TGA.ATT.GTA.ATA.CGA.CTC.ACT.ATA.GGG.AGG.CGG.TTT.TTT.TTT.TTT.TTT.TTT. TTT.TTT-3' 3. 1,7 M Trehalose (Sigma T-5251) 4. SuperScript II RT RNase H – reverse transcriptase, 200 U/µL (Invitrogen, 10.000 U, 18064-014) package contains: SuperScript II RT, 5× first strand buffer, 0,1M DTT 5. 10 mM dNTP-Mix (Amersham Pharmacia Biotech, 100 mM, 27-2035-01) 6. 5× second strand buffer: 100 mM Tris-HCl, pH 6,9 450 mM KCl 23 mM MgCl2 0,75 mM • -NAD + (Sigma, N-1636) 50 mM (NH4)2SO4 7. E. coli DNA-Ligase, 10 U/µL (Invitrogen, 100 U, 18052-019) 8. E. coli DNA-Polymerase I, 10 U/µL (Invitrogen, 1000 U, 18010-025) 9. E. coli RNase H, 2 U/µL (Invitrogen, 120 U, 18021-017) 10. DEPC-Wasser (Invitrogen, 10977-015) 11. Ethanol absolute 12. 3 M Sodium acetate, pH5,0 with glacial acetic acid 13. AmpliScribe T7 transcription kit (Epicentre technologies, AS3107) 14. QiaQuick PCR purification kit (Qiagen, 28106) 15. RNeasy mini kit (Qiagen, 74106) Protocol First strand synthesis Preparations: dissolve Trehalose at > 42 ° C completely under frequent mixing pre-warm a heating block to 75 °C thaw 5× first strand buffer, DTT and dNTP-Mix and place on ice Make up the following reaction: 5 µg Total RNA preparation 40U (1 µL) RNase Out adjust volume with RNase-free water to 4 µL, or dry out in a SpeedVac 200 ng (1 µL) HT7T-Primer 10,2 µmol (6 µL) 1,7 M Trehalose incubate for 5 min, 75 ° C then chill on ice and spin down 1/5 Vol. (4 µL) 5× first strand buffer 200 nmol (2 µL) 0,1 M DTT# 10 nmol (1 µL) 10 mM dNTP-Mix 1,7 µmol (1 µL) 1,7 M Trehalose 200 U (1 µL) SuperScript II RT transfer the mixture into 0.2 mL tubes and incubate as followed: 1. 5 min 37 °C 2. 5 min 45 °C 3. 2 min 60 °C 4. 2 min 55 °C 5. repeat 9 more times from step 3 6. 10 min 65 °C 7. keep at 4 °C Second strand synthesis: Make up the following reaction: complete first strand product (20 µL) 1/5 Vol. (33,4 µL) 5× second strand buffer 34 nmol (3,4 µL) 10 mM dNTP-Mix 10 U (1 µL) E. coli DNA-Ligase 40 U (4 µL) E. coli DNA-Polymerase I 2 U (1 µL) E. coli RNase H up to 166,6 µL (103,8 µL) water incubate for 3 h, 16 °C incubate for 30 min 65 °C keep at 4 °C or on ice Purification with QiaQuick PCR purification kit 1. add 7 µL 3 M Sodium acetate to the second strand product 2. add 5 Vol. Buffer PB (usually 868 µL) 3. mix well and incubate for 2 – 3 min on ice 4. apply half of the mixture onto a QiaQuick column 5. centrifuge at 9000 rpm, 15 s, RT 6. discard the filtrate, place the column back into the receiver tube 7. apply the remaining mixture onto the column 8. centrifuge at 9000 rpm, 15 s, RT 9. discard the filtrate, place the column back into the receiver tube 10. add 500 µL Buffer PE onto the column 11. centrifuge at 9000 rpm, 15 s, RT 12. discard the filtrate, place the column back into the receiver tube 13. add another 500 µL Buffer PE onto the column 14. centrifuge at 9000 rpm, 15 s, RT 15. discard the filtrate, place the column back into the receiver tube 16. centrifuge at maximum speed, 1 min, RT 17. transfer the column to a fresh 1.5 mL tube 18. add 45 µL 1/5 EB onto the column 19. incubate for 1 min, RT 20. centrifuge at 9000 rpm, 15 s, RT 21. add 35 µL water onto the column 22. incubate for 1 min RT 23. centrifuge at 9000 rpm, 15 s RT 24. dry the samples in a SpeedVac --> dried cDNA In-Vitro-Transcription Preparations: thaw the 10× T7 reaction buffer at > 42 ° C and dissolve completely by frequent mixing thaw 25 mM rNTP-Mix and mix well thaw 100 mM DTT and mix well Compose the following mixture: 8 µL water 1/10 Vol. (2 µL) 10× T7 reaction buffer 150 nmol (6 µL) 25 mM rNTP-Mix 200 nmol (2 µL) 100 mM DTT dissolve the dried cDNA in 18 µL of the mixture by vortexing and put on ice add 2 µL AmpliScribe T7 enzyme solution incubate for 3 h, 37 °C incubate for 10 min, 65 °C keep at 4 °C or on ice Purification with Qiagen RNeasy mini kit 1. add 6 µL 2-Mercaptoethanol per 1000 µL RLT 2. pipet 300 µL Ethanol absolute into a 1.5 mL tube and keep on ice 3. add 300 µL RLT + ME to the sample 4. transfer the mixture to the tube with ethanol and mix well by pipeting 5. apply the mixture to an RNeasy column 6. centrifuge 9000 rpm, 15 s, RT 7. discard the filtrate 8. add 600 µL RW1 onto the column 9. centrifuge 9000 rpm, 15 s, RT 10. discard the filtrate and receiver tube and place the column into a fresh receiver tube 11. add 500 µL RPE onto the column 12. centrifuge 9000 rpm, 15 s, RT 13. discard the filtrate 14. add another 500 µL RPE onto the column 15. centrifuge 9000 rpm, 15 s, RT 16. discard the filtrate 17. centrifuge at maximum speed, 1 min, RT 18. discard the receiver tube and place the column into a fresh RNase-free 1.5 mL tube 19. add 45 µL RNase-free water onto the column 20. incubate for 1 min, RT 21. centrifuge at 9000 rpm, 15 s, RT 22. incubate for 1 min, RT 23. centrifuge at 9000 rpm, 15 s, RT 24. take a 1:100 dilution of the sample for spectrophotometric measurement at 260 nm 25. determine the amount of RNA from the absorbance at 260 nm 26. samples can be stored a –20 °C --> cRNA Labeling Materials 1. RNase Out, 40 U/µL (Invitrogen, 10777-019) 2. Random-Nanomers N9, 1 µg/µL (Metabion) 3. SuperScript II RT RNase H – reverse transcriptase (Invitrogen, 18064-014) package contains: SuperScript II RT, 5× first strand buffer, 0,1M DTT 4. –C-dNTP-Mix: 10 mM dATP, 2 mM dCTP, 10 mM dGTP, 10 mM dTTP (Amersham Pharmacia Biotech, 272050, 272060, 272070, 272080) 5. Cy3-dCTP for Microarrays (Amersham Pharmacia Biotech, PA 53021) 6. Cy5-dCTP for Microarrays (Amersham Pharmacia Biotech, PA 55021) 7. 2,5 M NaOH 8. 2 M MOPS 9. 3 M Sodium acetate, pH 5,0 with glacial acetic acid 10. QiaQuick PCR Purification Kit (Qiagen, 28106) Protocol Preparations: pre-warm a heating block to 70 ° C thaw 5× first strand buffer, DTT und –C-dNTP-Mix and keep on ice don't work in bright light while usind Cy dyes! Make up the following reaction: 5 µg cRNA 20 U *) (0,5 µL *)) RNase Out adjust with RNase-free water to 8 µL; *) if the volume of cRNA exceeds 8,5 µL, add 40 U (1 µL) RNase Out and dry out in a SpeedVac 2 µg (2 µL) Random-Nanomers N9 incubate for 10 min, 70 ° C, chill on ice and spin down 1/5 Vol. (4 µL) 5× first strand buffer 2 nmol dCTP, 10 nmol dDTP (1 µL) –C-dNTP 200 nmol (2 µL) 0,1 M DTT 1,5 nmol (1,5 µL) Cy5-dCTP 200 U (1 µL) SuperScript II RT incubate for 2½ h 42 °C chill on ice or go to 4 °C add 2 µL 2,5 M NaOH incubate for EXACTLY 15 min, 37 ° C add 10 µL 2 M MOPS Purification with QIAquick PCR Purification Kit 1. add 5 µL 3 M Sodium acetate, pH5 to the sample 2. add 5 Vol. Puffer PB zugeben (usually 185 µL) 3. mix well 4. pipet the mixture onto a QiaQuick column 5. centrifuge at 9000 rpm, 15 s, RT 6. discard the filtrate 7. add 500 µL PE onto the column 8. centrifuge at 9000 rpm, 15 s, RT 9. discard the filtrate 10. add another 500 µL onto the column 11. centrifuge at 9000 rpm, 15 s, RT 12. discard the filtrate 13. centrifuge at maximum speed, 1 min 14. discard the filtrate and receiver tube 15. transfer the column into a fresh 1.5 mL tube 16. add 50 µL 1/5 EB onto the column 17. incubate for 1 min, RT 18. centrifuge at 9000 rpm, 15 s, RT 19. if any remains of dye are visible on the membrane, add another 50 µL onto the column 20. incubate for 1 min, RT 21. centrifuge at 9000 rpm, 15 s, RT 22. dry out in a SpeedVac (darken the SpeedVac with aluminum foil) 23. store the samples at –20 °C in the dark --> Cy5-labeled target
Parameters
Amount of nucleic acid labeled, Amplification, Label used
Links
All experiments using protocol P-MEXP-1959: (E-CAGE-10, E-CAGE-5, E-CAGE-6, E-CAGE-7, E-CAGE-8, E-CAGE-9)