PENN SCAP-T Protocols

CM aRNA Protocol

Version 1

Universisty of Pennsylvania

 

I. First Round, Second Strand cDNA synthesis ON ICE

II. First Round, IVT using Ambion MEGAscript T7 Kit AT ROOM TEMPERATURE

III. Second Round, First Strand synthesis AT ROOM TEMPERATURE

IV. Second Round, Second strand cDNA synthesis ON ICE

V. Second Round, IVT using Ambion MEGAscript T7 Kit AT ROOM TEMPERATURE

 

 

 

I. First Round, Second Strand cDNA synthesis ON ICE
  1. 20 μl first strand cDNA sample
    7.5 μl 5X Second strand buffer
    0.75 μl dNTP mix (2.5mM each)
    1 μl DNA polymerase I (10U/μl)
    0.25ul water
    0.25 μl RNase H (2U/μl)
  2. Mix thoroughly by pipetting and spin briefly
  3. Incubate 2 hours at 16°C (Sean’s bench)
  4. Add 1 μl T4 DNA polymerase (5 U/μl)
  5. Mix by pipetting and spin briefly
  6. Incubate 10 more minutes at 16°C
  7. Clean up reaction with Minelute kit
  8. Concentrate sample to ~2-4 uL using Speedvac

II. First Round, IVT using Ambion MEGAscript T7 Kit AT ROOM TEMPERATURE
  1. Bring cDNA volume up to 4 uL with Minelute elution buffer
  2. Add 8 μl NTP mix (18.75 mM each)-on ice 2μl 10X reaction buffer-RT while assembling rxn
  3. Mix well to fully resuspend pellet and add 2 μl 10X enzyme mix (on ice)
  4. Incubate in thermocycler w/ heated lid Program: 37° C 14 hours 4°C hold
  5. Clean up reaction with MEGAclear kit
  6. Concentrate sample w/ EtOH precip

III. Second Round, First Strand synthesis AT ROOM TEMPERATURE
  1. Bring aRNA volume to 4 uL with MEGAclear elution buffer
  2. + 1 μl Random primers (0.05 mg/ml)
  3. Heat at 70°C for 10 minutes
  4. Immediately place on ice for at least 2 minutes
  5. Add 2 μl 5X First strand buffer
    1 μl DTT (100mM)
    0.5 μl dNTP’s (2.5 mM each)
    0.5 μl RNasin
    1 μl Superscript III
  6. Allow to sit at RT for 10 minuntes
  7. Incubate 30 minutes at 42°C
  8. Heat 5 minutes at 95°C to denature
  9. Place on ice for at lease 2 minutes
  10. Spin briefly

IV. Second Round, Second strand cDNA synthesis ON ICE

  1. Add 2 μl dT-T7 oligo (10 ng/μl)
  2. Heat for 6 minutes at 70°C
  3. Immediately place on ice for at least 2 minutes
  4. Spin briefly
  5. Add 43.5 μl DEPC water
    15 μl 5X Second strand buffer
    1.5 μl dNTP mix (2.5 mM each)
    2 μl DNA polymerase I
  6. Mix thoroughly by pipetting and spin briefly
  7. Incubate 2 hours at 16°C
  8. Add 2 μl T4 DNA polymerase (5U/μl)
  9. Mix thoroughly by pipetting and spin briefly
  10. Incubate 10 more minutes at 16°C
  11. Clean up reaction with Minelute kit
  12. Concentrate sample to ~2-4 uL using Speedvac

V. Second Round, IVT using Ambion MEGAscript T7 Kit AT ROOM TEMPERATURE

  1. Bring cDNA sample to 4 uL with MEGAclear elution buffer
  2. Add 8 μl NTP mix (18.75 mM each)
    2 μl 10X reaction buffer
  3. Mix well to fully resuspend pellet and add 2 μl 10X enzyme mix
  4. Incubate 14 hours in thermocycler with heated lid
    Program: 37°C 14 hours
    4° C hold
  5. Clean up reaction with MEGAclear kit
  6. Concentrate sample w/ EtOH precip

 

Repeat sections III-V for further rounds of amplification
For production of microarray probes: Third round IVT with
Illumina TotalPrep RNA Amplification Kit

Notes: save 3μl after 2nd and 3rd rd minelute cleanup
Add in control RNA

 

   

 

 

 

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