RT reactions

background prep for RT reactions

• resuspended common-P1 at 1000 uM concentration
• 405 primer from Invitrogen not arrived yet
• compare superscriptII to superscriptIII in yield
• superscriptII protocol
• superscriptIII protocol
• 85 bp 1000 ng/uL ~ 35 pmol per uL = 350 pmol in 10 uL
• dilute common-P1 working stock 1:10 for 100 uM (100 pmol per uL)

conditions

• superscriptII, 150 pmol primer, 5 ug RNA, 42C. (20 uL): G2-G4
• superscriptIII, 150 pmol primer, 5 ug RNA, 55C. (20 uL): G2-G4
• 1/4 superscriptIII, 150 pmol primer, 5 ug RNA, 55C. (20 uL): G2-G4
• superscriptIII, 450 pmol primer, 10 ug RNA, 55C (20 uL): G2-G6 + G8

Master mixes and protocol.

• 18 reactions
• buffer mix: 4 uL FS buffer, 2 uL .1M DTT, 1 uL RNasin
• 19x buffer master
  • 76 uL FS buffer
  • 38 uL .1M DTT
  • 19 uL RNasin
• 150 pmol primer, 5 ug RNA = 1 uL dNTPs + 1.5 uL common-P1 + 5uL ddH2O + 5.5 uL RNA template.
• 13x dNTP / primer master for 150 pmol primer, 5 ug RNA
  • 13 uL dNTPs
  • 19.5uL common-P1
  • 52 uL ddH2O
• For the 12 (150 pmol + 5ug): Add 7 uL dNTP-primer to 5.5 uL of each RNA template.
• 7x dNTP / primer master for 450 pmol primer, 10 ug RNA
  • 7 uL dNTPs
  • 1.5 uL of 1000 uM primer
• For the 6 (450 pmol + 10 ug): Add 1.2 uL of this master to 10.5 uL of each RNA template
• Heat Nucleotide/primer/RNA mixes at 65C 5 min. Chill on ice.
• to 4 + 6 full strength SSIII reactions, add 10 uL SSIII enzyme to 70 uL of buffer. Add 8 uL per reaction
• to the 4 quarter strength SSIII reactions, add 1 uL of SSIII enzyme to 30 uL of buffer. Add 7.25 uL per reaction
• Heat all SSIII reactions at 55C for 1.5 hours, then 70C then 4C hold.
• Add 7 uL buffer mix to each of the 4 SSII samples
• heat at 55C for 2 min
• Add 1 uL SSII enzyme per reaction for the 4 SSII samples.
• Heat SSII reaction at 42C for 1.5 hours, then 70C then 4C hold.

superscriptII reactions (G2-G4)

• buffer mix: 4 uL FS buffer, 2 uL .1M DTT, 1 uL RNasin
• 5x buffer master
  • 20 uL FS buffer
  • 10 uL .1M DTT
  • 5 uL RNasin
• 1 uL dNTPs + 1.5 uL common-P1 + 5 uL RNA template.
• dNTP / primer master
  • 5 uL dNTPs
  • 7.5uL common-P1
  • 5.5 uL ddH2O
• Add 7.5uL dNTP-primer to 5 uL of each RNA template.
• Heat at 65C 5 min
• Add 7 uL buffer mix.
• heat to 42C 2 min
• Add 1 uL enzyme per reaction

Results:

concentrations: