Setting up quantitative real-time PCR qRT-PCR: Difference between revisions
Jump to navigation
Jump to search
No edit summary |
No edit summary |
||
| Line 9: | Line 9: | ||
== qRT-PCR == | == qRT-PCR == | ||
{{Pipetten-Checklist-8step | |||
|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}} | |||
Revision as of 11:17, 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
| ☐ | 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 |