Matt:LabNotes/2014-6-18
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Repeat Top48 Hexamer RT Primer in vitro Validation[edit]
Procedure[edit]
First Strand Synthesis[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- Add 2 ul HMW buffer to each PCR tube (8 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 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
Adapter PCR[edit]
- Finish first round PCR master mix (per reaction):
- 15 ul 2X KAPA fast SYBR.
- 0.6 ul Adapter 1.
- 1.4 ul H2O.
- Add 0.6 of barcode adapters to each sample
- Random Primer Mix: 26
- Oligo (dT)23 VN: 27
- FISSEQ_RT: 28
- Top48 Hexamer: 29
- NTC: 30
- No Enzyme: 31
- NxtraPosControl (gDNA): 32
- NxtraNegControl (H2O): 33
- NxtraPCRControl (H2O): 34
- Add 17 ul of first round PCR master mix to each sample
- Incubate in thermocycler:
- 95 degC for 1 min.
- 7 cycles:
- 95 degC for 10 sec.
- 62 degC for 15 sec.
- 72 degC for 45 sec.
- Finish second round PCR master mix:
- 35 ul KAPA fast SYBR
- 2 ul Primer 1
- 2 ul Primer 2
- 31 ul H2O
- Add 70 ul of second round PCR master mix to each sample.
- PCR program is as follows:
- 95 degC for 1 min.
- 10 cycles
- 95 degC for 10 sec.
- 60 degC for 15 sec.
- 72 degC for 45 sec.
- Incubate in thermocycler an additional 3 min @ 72 degC for final extension.
File:06202014 Top48HexPrimer NxtraPCRtrial2.JPG
- Run 5 ul sample on PAGE gel with 3 ul 6X loading dye to identify positives.
- 0.5 ul low mass ladder (Invitrogen). Equalize volume with TBE.
- 25 min @ 250 V.
- Stain 5-10 min in ~100 ml 1X TBE with 6 ul SYBR Safe.
File:2014-06-20 RTPrimerinvitroTest Nextera trial2.jpg
- Add 100 ul magnetic beads to each positive sample. Mix by pipetting and incubate 8 minutes off magnet
- Place on magnet 5 minutes, then remove and discard supernatant
- On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing
- Let dry 5 minutes
- Resuspend beads off magnet with 20 ul H2O and incubate 2 minutes, then return to magnet for 1 minute.
- Extract supernatant and transfer to 0.2 ml tube
- Continued on: Matt:LabNotes/2014-6-23