Matt:LabNotes/2014-9-25: Difference between revisions

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**NoEnzyme had no amplification which is GOOD unlike last time, indicates all DNA was removed from UHRR
**NoEnzyme had no amplification which is GOOD unlike last time, indicates all DNA was removed from UHRR
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# 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 | 650px]]
# Add 100 ul magnetic beads to each positive sample. Mix by pipetting and incubate 8 minutes off magnet
# 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
# Place on magnet 5 minutes, then remove and discard supernatant
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# Extract supernatant and transfer to 0.2 ml tube
# Extract supernatant and transfer to 0.2 ml tube


====Gel Size Selection====
*Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye
*Samples: 5ul Sample + 2ul Loading Dye
*Gel 1:
[[File:2014-06-24_SizeSelection_Gel1.jpg | 450px]]
*Gel 2:
[[File:2014-06-24_SizeSelection_Gel2.jpg | 450px]]
#Selected size: 200-800bp but went a little conservative so more like 250bp-750bp
#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 45-60 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 6 tubes at -80C for 20 minutes
#Spun 6 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 6 tubes with 10ul H2O
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Revision as of 23:32, 29 September 2014

Repeat Top48 RT Primer in vitro Validation after DNA removal from UHRR

  • Must first remove dsDNA from UHRR since there was contamination last time

Purify UHRR of DNA

  • Use Zymo DNA-Free RNA Kit
  • Sample: 4ul of UHRR (1.3 ug/ul)

DNase I Digest

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

  • 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

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
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

  • 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

Nextera Tagmentation

  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

  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