5.5 RT-PCR

Prepare cDNA, and use it as a template for RT-PCR.

4 RT reactions: For each RNA sample (two tissues or two conditions): one +RT and one RT

2015

  • Each group can do up to 9 rxns (3x3x3), with some doing 8 (2x4) or as few ask 6 (2x3). This way there will be replicates

  • We will give them 12 cDNA's (2 tissues x 2 conditions x 3 replicates)

  • 9 rxns x 19 groups + 12 salt rxns by the TA's = 183. Buy 4 50-rxn RNEasy kits from Qiagen

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

RT Reagents

  • 1 tube per group + 5 extra = 15 tubes of each reagent (except RNaseOUT & superscript)

  • dispense

µL per group reagent actual need (µL) 15 aliquots (µL) notes
8 Oligo dT 50 µM 1 1 x 4 120
8 10 mM dNTP mix 2 1 X 4 120
60 RNAse-free water 13 x 4 900 aliquot once for both RT and qPCR
20 5X 1st strand buffer 4 x 4 300 comes with Superscript
8 0.1M DTT 1 x 4 120 comes with Superscript
(4 ) RNaseOUT 3 1 x 4 60 Dispensed from freezer box during lab
(2 ) superscript III 4 1 x 2, 30 dispensed from freezer box during lab

PCR Reagents

1 tube per group + 5 extra (except Taq and primers) dispense per group actual need

µL per group reagent actual need (µL) 15 aliquots (µL) notes
550 Sterile ddH2O 360 8250
75 10× Ex-Taq Buffer 50 1275
50 2.5 mM dNTP Mix 40 850
15 GSP1 12.5 μM 10 check their supply (or ask at the beginning of class)
15 GSP2 12.5 μM 10 check their supply (or ask at the beginning of class)
2 cDNA +/- Fe, each 2 30 dispense during lab
10 Ex-Taq Polymerase 1U/μL 10 150 dispensed during lab - can dilute to 0.8U/µL

  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 

AttachmentSize
rnase_out.pdf71.72 KB
superscriptiii_man.pdf254.7 KB