T7 in vitro Tx

Library2, samples G1-8

skip sample G1, no DNA recovery

NEB

1. For each sub-library, mix the following in a single PCR tube or well of a PCR strip or plate
   • 8 uL DNA template + 2uL ddH2o
   • 1.5 uL 10X T7 Reaction Buffer
   • 1.5 uL 100 mM ATP (or 6uL 25mM rNTPs)
   • 1.5 uL 100 mM CTP
   • 1.5 uL 100 mM GTP
   • 1.5 uL 100 mM UTP
   • 1 uL Murine RNase Inhibitor
   1. Mix well via pipetting and add
   • 1.5 uL T7 RNA polymerase mix
   1. Mix well via pipetting
   2. Incubate at 37C on a thermocycler for 16 hours

NEB Master Mix

• 48 uL 25 mM rNTPs
• 8 uL RNasin
• 12 uL 10x T7 buffer
• 12 uL T7 RNA pol mix
• 16 uL ddH2O
• 12 uL to each well + 8 uL DNA template

Promega T7

1. for a 40 uL reaction:
   • 4 uL DNA template (used most of template for other reactions)
   • 8 uL 5x Reaction buffer
   • 4 uL DTT
   • 4 uL RNasin
   • 4 uL 25 mM rNTPs
   • 0.5 uL T7 pol (80U/uL)
   • 15.5 ddH2O

* Mix well, incubate at 37C on a thermocycler for 16 hours

T7 Master Mix

• 64 uL 5x Reaction buffer
• 32 uL DTT
• 32 uL RNasin
• 16 uL 25 mM rNTPs (darn this is completely wrong). added an extra 3×8 =24 per well
• 86+36 ddH2O (also wrong, should have pre-calculated).