Matt:LabNotes/2014-6-18
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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
Purification & Measure Concentration
- Qiagen PCR Purification
- Elute with 30ul
- Nanodrop quant:
- Random Primer Mix: 15.2 ng/ul
- Oligo (dT)23 VN: 23.6 ng/ul
- FISSEQ_RT: 9.7 ng/ul
- Top48 Hexamer: 24.3 ng/ul
- NTC: 14.5 ng/ul
- No Enzyme: 25.0 ng/ul
- Negative controls (NTC & No Enzyme) did not show significantly less DNA, could be RNA absorbing light and "fooling" the Nanodrop
- Actual concentrations probably much less since previous comparison with Qubit showed 25x less
Nextera Tagmentation
- Add 2 ul HMW buffer to each PCR tube (7 total)
- Transfer 7ul of each sample to tubes
- Random Primer Mix: 15.2 ng/ul
- Oligo (dT)23 VN: 23.6 ng/ul
- FISSEQ_RT: 9.7 ng/ul
- Top48 Hexamer: 24.3 ng/ul
- NTC: 14.5 ng/ul
- No Enzyme: 25.0 ng/ul
- NxtraPosControl (gDNA): 0.803 ng/ul
- NxtraNegControl (H2O): 0 ng/ul
- Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5.5 minutes at 55 degC
- Dilute Qiagen protease 1:100 in H2O. Add 1 ul to each sample and incubate 10 minutes at 50 degC, followed by 20 minutes at 70 degC
- Make the master mix for Exo- treatment: 1 ul Exo- and 0.4 ul dNTP per reaction
- Add 1.4 ul Exo- mix to each sample and incubate 15 minutes at 37 degC, followed by 20 minutes at 75 degC