RT, qPCR

RT, qPCR kdorfman Tue, 04/26/2022 - 14:16

Separate RT & qPCR

RT 10 groups @ 6 rxns 2 groups @ 8 rxns

Equipment

Hot blocks at

  • 50°C (2 dry baths - this is the long step and there are at least 4x as many tubes as groups),
  • 65°C
  • 70°C
    reagent uL/rxn uL/8 rxn (+ xtra) uL/class notes
    Oligo dT 50 µM 1 1 15 180
    10 mM dNTP mix 2 1 15 180
    RNAse-free water 11 + 40 60 720 aliquot once for both RT and qPCR
    5X 1st strand buffer 4 60 720 comes with Superscript
    0.1M DTT 1 15 125 1500 | comes with Superscript
    RNaseOUT 3 1 8 96 Dispensed from freezer box during lab
    Superscript 4 1 8 96 Dispensed from freezer box during lab

864 rxns @ 10uL

(24 x 3 rxns per group x 12 groups)

Reagent uL per rxn uL per 75 rxns
2x SYBR Green master mix5 5 uL 375 uL
water <5 uL 375 uL
forward primer 0.5 uL
reverse primer 0.5 uL
cDNA ~1 uL

  1. Fisher FERSO131 Oligo dT 100 µM (=0.5µg/µL) 60 µL @ ~$50. Dilute to 50µM!! (for emergencies: 50 µM oligo(dT) Fisher stockroom: C1101 0.5µg/µL x 40µL = 20µg) - Order T(18) from Invitrogen??? ↩︎

  2. 14 µL each + 84 µL RNase-free water OR Fisher FERR0192 ↩︎

  3. Invitrogen 10777019 RNAse out 5KU @ $143 ↩︎

  4. superscript III 200U/µL Invitrogen 18080044 10KU @ $259

    qPCR ↩︎

  5. Thermo Fisher 4385612 ↩︎