Setting up quantitative real-time PCR qRT-PCR: Difference between revisions
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== cDNA synthesis from RNA == | |||
{{Pipetten-Checklist-8step | {{Pipetten-Checklist-8step | ||
|schritt1=5X RT buffer|menge1=4 µL | |schritt1=5X RT buffer|menge1=4 µL | ||
}} | |schritt3=reverse transcriptase|menge3=1 µL|menge2=2 µL|schritt2=dNTP-Mix|schritt4=random hexamer primer (160 µM), primer #312|menge4=1.25 µL|schritt5=oligo dT18 (100 µM); primer #945|menge5=0.1 µL|schritt6=RNA|menge6=1-2 µg|schritt7=RNA Inhibitor|menge7=0.5 µL|schritt8=–|menge8=–|zusatz1=RNAse-free dd H2O|titel=Reaction mix per sample|gesamt=up to 20 µL}} | ||
* Incubation of sample for 1 h at 37°C | |||
* Heat-inactivation of enzyme at 70°C for 15 min | |||
== qRT-PCR == | |||
{{Pipetten-Checklist-8step | |||
|schritt1=H<sub>2</sub>O|menge1=18.3 µL | |||
|schritt3=Primer 1|menge3=0.5 μL (10 pmol)|menge2=2.5 µL|schritt2=10x Taq/Pfu buffer|schritt4=Primer 2|menge4=0.5 μL (10 pmol)|schritt5=Template|menge5=2 μL (~10 ng)|schritt6=dNTPs|menge6=0.2 µL|schritt7=SYBR Green|menge7=0.5 µL|schritt8=Taq Polymerase|menge8=0.5 µL|zusatz1=Total|titel=Reaction mix per sample|gesamt=25 µL}} | |||
=== Important notes: === | |||
* optimized for AppliChem SYBR Green CAS# A8511 | |||
* SYBR Green in higher concentration inhibits polymerase activity | |||
Latest revision as of 11:20, 30 May 2025
cDNA synthesis from RNA
✓ Reaction mix per sample
| ☐ | 5X RT buffer | 4 µL |
| ☐ | dNTP-Mix | 2 µL |
| ☐ | reverse transcriptase | 1 µL |
| ☐ | random hexamer primer (160 µM), primer #312 | 1.25 µL |
| ☐ | oligo dT18 (100 µM); primer #945 | 0.1 µL |
| ☐ | RNA | 1-2 µg |
| ☐ | RNA Inhibitor | 0.5 µL |
| ☐ | – | – |
| ☐ | RNAse-free dd H2O | up to 20 µL |
- Incubation of sample for 1 h at 37°C
- Heat-inactivation of enzyme at 70°C for 15 min
qRT-PCR
✓ Reaction mix per sample
| ☐ | H2O | 18.3 µL |
| ☐ | 10x Taq/Pfu buffer | 2.5 µL |
| ☐ | Primer 1 | 0.5 μL (10 pmol) |
| ☐ | Primer 2 | 0.5 μL (10 pmol) |
| ☐ | Template | 2 μL (~10 ng) |
| ☐ | dNTPs | 0.2 µL |
| ☐ | SYBR Green | 0.5 µL |
| ☐ | Taq Polymerase | 0.5 µL |
| ☐ | Total | 25 µL |
Important notes:
- optimized for AppliChem SYBR Green CAS# A8511
- SYBR Green in higher concentration inhibits polymerase activity