Matt:LabNotes/2014-6-9
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Repeat Top48 Hexamer RT Primer in vitro Validation
- Last time negative control
Procedure
First Strand Synthesis
Component | Pos Ctrl | Neg Ctrl | Exp |
25mM dNTP | 1 | 1 | 1 |
1.3ug/ul UHRR | 0.5 | 0.5 | 0.5 |
100uM RanHex Primer | 0.5 | 0.5 | 0 |
100uM Top48 Primer | 0 | 0 | 0.5 |
H2O | 7 | 7 | 7 |
- Heat reaction mix (above) to 65C for 5min
- Add 1ul 10X M-MuLV RT Buffer (total 10ul volume)
- Incubate 25C for 10min
- Add 1ul M-MuLV Reverse Transcriptase to Pos Ctrl and Exp ONLY (1ul H2O for Neg Ctrl)
- Incubate 42C for 60min
- Inactivate by heating to 85C for 10min
Second Strand Synthesis
- Add 57ul H2O
- Add 8ul 10X Second Strand Synthesis Buffer
- Add 4ul Second Strand Enzyme
- Mix by pipetting
- Incubate 16C for 2.5 hours
- Purify with magnetic bead (120ul beads to 80ul reaction)
- Elute with 25ul H2O