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.