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]

  1. Add 2 ul HMW buffer to each PCR tube (8 total)
  2. 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)
  3. Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5 minutes at 55 degC
  4. 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
  5. Make the master mix for Exo- treatment: 1 ul Exo- and 0.4 ul dNTP per reaction
  6. 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]

  1. Finish first round PCR master mix (per reaction):
    1. 15 ul 2X KAPA fast SYBR.
    2. 0.6 ul Adapter 1.
    3. 1.4 ul H2O.
  2. 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
  3. Add 17 ul of first round PCR master mix to each sample
  4. Incubate in thermocycler:
    1. 95 degC for 1 min.
    2. 7 cycles:
      1. 95 degC for 10 sec.
      2. 62 degC for 15 sec.
      3. 72 degC for 45 sec.
  5. Finish second round PCR master mix:
    1. 35 ul KAPA fast SYBR
    2. 2 ul Primer 1
    3. 2 ul Primer 2
    4. 31 ul H2O
  6. Add 70 ul of second round PCR master mix to each sample.
  7. PCR program is as follows:
    1. 95 degC for 1 min.
    2. 10 cycles
      1. 95 degC for 10 sec.
      2. 60 degC for 15 sec.
      3. 72 degC for 45 sec.
  8. 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]

  1. Add 100 ul magnetic beads to each positive sample (samples 1-4). Mix by pipetting and incubate 8 minutes off magnet
  2. Place on magnet 5 minutes, then remove and discard supernatant
  3. On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing
  4. Let dry 5 minutes
  5. Resuspend beads off magnet with 20 ul H2O and incubate 2 minutes, then return to magnet for 1 minute.
  6. 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

  1. Selected size: 200-800bp
  2. 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.
  3. Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
  4. Added 450 ul of 1X TE buffer
  5. Vortexed for 60 min at 37 C in incubator
  6. Centrifuged at 15,000 rpm for 3 min at RT
  7. Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
  8. Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  9. Transfered spnt to fresh 1.5 mL tube (volume ~ 400ul/ tube)
  10. Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  11. Vortexed and placed the 4 tubes at -80C for overnight
  12. Spun 4 tubes in -80C at 10,000rpm at 4C for 30min
  13. Discard supernatant and add 650ul 75% EtOH to wash and spin at 14,000rpm at 4C for 5 min
  14. Discard supernatant and let dry in hood 10 min
  15. Resuspended each of the 4 tubes with 10ul H2O