Matt:LabNotes/2014-9-25
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Repeat Top48 RT Primer in vitro Validation after DNA removal from UHRR[edit]
- Must first remove dsDNA from UHRR since there was contamination last time
Purify UHRR of DNA[edit]
- Use Zymo DNA-Free RNA Kit
- Sample: 4ul of UHRR (1.3 ug/ul)
DNase I Digest[edit]
Components | Volume |
UHRR (1.3ug/ul) | 4 |
10X DNase I Buffer | 5 |
DNase I | 2 |
H2O | 39 |
Total | 50 |
- Mix and incubate at 37C for 30min
Purify[edit]
- Add 100ul RNA Binding Buffer and mix
- Add 150ul 100% EtOH and mix
- Transfer to Zymo-Spin IC Column in Collection Tube
- Centrifuge at 13,000rcf for 1min and discard flow through
- Add 400ul RNA Prep Buffer
- Centrifuge at 13,000rcf for 1min and discard flow through
- Add 800ul RNA Wash Buffer
- Centrifuge at 13,000rcf for 30sec and discard flow through
- Add 400ul RNA Wash Buffer
- Centrifuge at 13,000rcf for 30sec and discard flow through
- Centrifuge at 13,000rcf for 2min and discard flow through
- Transfer column to 1.5ul tube and add 8ul H2O and let stand for 1min
- Centrifuge at 10,000rcf for 30sec
- Store at -20C
Reverse Transcription & Second Strand Synthesis[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 |
Purified UHRR | 1 | Purified UHRR | 1 | Purified UHRR | 1 | Purified UHRR | 1 | Purified UHRR | 0 | Purified UHRR | 1 |
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 | H2O | 5 | H2O | 6 | H2O | 6 | H2O | 6 | H2O | 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
Nextera Tagmentation[edit]
- Add 2 ul HMW buffer to each PCR tube (8 total)
- Transfer 7ul of each sample to tubes
- Random Primer Mix
- Oligo (dT)23 VN
- FISSEQ_RT
- Top48 Hexamer
- NTC
- No Enzyme
- NxtraPosControl (gDNA): 0.803 ng/ul
- NxtraNegControl (H2O)
- 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): 33
- NxtraNegControl (H2O): 34
- NxtraPCRControl (H2O): 35
- 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:20140929 RTprimertestNxtraPCR.JPG
- RanHex, dT, FISSEQ_RT, Top48, and NxtraPosControl (gDNA) all had amplification as expected
- NTC, NoEnzyme, NxtraNegControl, and NxtraPCRControl all had no amplification as expected
- NoEnzyme had no amplification which is GOOD unlike last time, indicates all DNA was removed from UHRR
Bead Purification[edit]
- Add 100 ul magnetic beads to each positive sample (samples 1-4). 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
Gel Size Selection[edit]
- Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye
- Samples: 5ul Sample + 2ul Loading Dye
- Gel:
File:2014-10-03 RTprimerTestSizeSelect.jpg
- Selected size: 200-800bp
- Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
- Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
- Added 450 ul of 1X TE buffer
- Vortexed for 60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
- Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transfered spnt to fresh 1.5 mL tube (volume ~ 400ul/ tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
- Vortexed and placed the 4 tubes at -80C for overnight
- Spun 4 tubes in -80C at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 75% EtOH to wash and spin at 14,000rpm at 4C for 5 min
- Discard supernatant and let dry in hood 10 min
- Resuspended each of the 4 tubes with 10ul H2O