Matt:LabNotes/2014-6-18: Difference between revisions
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>Mzcai (Created page with "==Repeat Top48 Hexamer RT Primer in vitro Validation== ===Procedure=== ====First Strand Synthesis==== *Mix RNA in Primer and H2O *Denature for 5 minutes at 70C **Put on ice ...") |
>Mzcai mNo edit summary |
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| M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||H2O||2 | | M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||H2O||2 | ||
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====Second Strand Synthesis==== | |||
*Add 48 ul H20 | |||
*Add 8ul 10x Second strand synthesis reaction buffer | |||
*Add 4ul Second strand synthesis enzyme mix | |||
*Mix by pipetting | |||
*Incubate for 2.5 hours at 16C |
Revision as of 23:01, 18 June 2014
Repeat Top48 Hexamer RT Primer in vitro Validation
Procedure
First Strand Synthesis
- Mix RNA in Primer and H2O
- Denature for 5 minutes at 70C
- Put on ice right after
- Add Reaction Mix and Enzyme
- Incubate for 5 min at 25C
- Incubate for 1 hour at 42C
- Inactivate enzyme for 5 min at 80C
Random Primer Mix | ' | Oligo d(T)23 VN | ' | FISSEQ_RT | ' | Top48 Hexamer | ' | NTC | ' | No Enzyme | ' |
Components | Volume | Components | Volume | Components | Volume | Components | Volume | Components | Volume | Components | Volume |
1.3ug/ul UHRR | 0.5 | 1.3ug/ul UHRR | 0.5 | 1.3ug/ul UHRR | 0.5 | 1.3ug/ul UHRR | 0.5 | 1.3ug/ul UHRR | 0 | 1.3ug/ul UHRR | 0.5 |
Random Primer Mix (60uM) | 2 | Oligo d(T)23 VN (50uM) | 2 | FISSEQ_RT (100uM) | 1 | Top48 Hexamer (100uM) | 1 | Random Primer Mix (60uM) | 2 | Random Primer Mix (60uM) | 2 |
H2O | 5.5 | H2O | 5.5 | H2O | 6.5 | H2O | 6.5 | H2O | 6 | H2O | 5.5 |
Denature Step | Denature Step | Denature Step | Denature Step | Denature Step | Denature Step | ||||||
M-MuLV Reaction Mix | 10 | M-MuLV Reaction Mix | 10 | M-MuLV Reaction Mix | 10 | M-MuLV Reaction Mix | 10 | M-MuLV Reaction Mix | 10 | M-MuLV Reaction Mix | 10 |
M-MuLV Enzyme | 2 | M-MuLV Enzyme | 2 | M-MuLV Enzyme | 2 | M-MuLV Enzyme | 2 | M-MuLV Enzyme | 2 | H2O | 2 |
Second Strand Synthesis
- Add 48 ul H20
- Add 8ul 10x Second strand synthesis reaction buffer
- Add 4ul Second strand synthesis enzyme mix
- Mix by pipetting
- Incubate for 2.5 hours at 16C