AlanFung:LabNotes/Rolony/2013-11-5

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Extraction of RNA from PGP1F cells using Zymo RNA MiniPrep[edit]

  • Start off with 1 10cm PGP1F cells, final elute with 25ul ddH2O
  • Yield 476ng/ul, A260/280 2.0

RT w random hexamer[edit]

  • Dilute FISSEQ_RT primer from 100uM to 10uM
  • UV mineral oil, 0.2ml tube in hood
Content Volume
25 mM dNTP (Enzymatics) 1
2µg Total RNA 4.2
10uM FISSEQ_RT (final 10 µg/mL) 1.34
4mM aminoallyl dUTP (Anaspec) 1
  • Heat reaction for 5 minutes at 65°C.
  • Spin briefly (5 sec) to pull down condensate and place immediately on ice.
Content Volume
RNase Inhibitor (Enzymatics) 0.4
10x MMLV reaction buffer 1
ddh2o 1.06
  • 2 minutes @ 25°C
  • Add 1 µL (200 units) M-MuLV Reverse Transcriptase and mix by gently pipetting sample
  • Add mineral oil to prevent evaporation
  • 25C 10min
  • Incubate at 42°C overnight
  • Inactivate enzyme at 85°C for 10 minutes.
  • Store products at -20°C
  • save 1ul for denaturing gel

Purification with Minelute[edit]

  • add 70ul DNA binding buffer to cDNA/RNA hybrid
  • load mixture into column
  • spin @ 10,000 rpm for 30 sec
  • Discard flow through
  • wash with 200ul wash buffer
  • place into clean 1.5ml tube and elute with 25ul elution buffer
  • Quick denature 3ul at 95C for 1 min transfer to ice
  • Nanodrop
  • 58.3ng/ul 260/280: 1.66 260/230: 1.45
  • Yield 1.46ug cDNA/RNA Hybrid

Degrade Residual RNA[edit]

  • 58.3ng/ul * 22ul =1282.6ng or 1.28ug need only 0.01U of RiboShredder to degrade RNA, use 1U to drive reaction to completetion
  • Add 1ul RiboShreder + 5ul of RNase H + 3.1ul RNase H Buffer to 22ul of cdna/rna hybrid
  • Incubate at 37C for 1 hour

Purify cDNA[edit]

  • add 220ul DNA binding buffer to cDNA
  • load mixture into column
  • spin @ 10,000 rpm for 30 sec
  • Discard flow through
  • wash with 200ul wash buffer
  • place into clean 1.5ml tube and elute with 25ul elution buffer
  • Nanodrop 5.8ng/ul 260/280:1.18 260/230:1.21
  • Yield 145ng

Denaturing gel to verify RNA/cDNA size[edit]

  • I will be running a denaturing urea gel since RNA is susceptible to intra-strand H-bonding and 2nd structure will affect its migration
  • DNA Ladder_RNA/RNAcDNA/RNAcDNA_purified/cDNA

File:ZhangLab 2 2013-11-08 16hr 43min.jpg

  • Most of the fragments are gone after the RNAse digestion, I am afraid that the RT did not complete and the RNAse might have degrade the RNA template.