5.5 RT-PCR
5.5 RT-PCR margaret Wed, 11/16/2011 - 18:14Alternate kit to try AzuraQuant cDNA synthesis kit 25 rxns $89; 100 rxns $299
** Should this be done in the thermocycler?**
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 |
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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??? ↩︎
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14 µL each + 84 µL RNase-free water OR Fisher FERR0192 ↩︎
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Invitrogen 10777019 RNAse out 5KU @ $143 ↩︎
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superscript III 200U/µL Invitrogen 18080044 10KU @ $259 ↩︎