Ho Suk:cDNA Rolony construction
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In Situ Whole Transcriptome Amplification From the Cell Monolayer[edit]
- Add 10% formalin in PBS for 15 min at RT.
- Incubate in 0.25% Triton-X100 in DEPC-PBS for 15 min, OR wash with ethanol for 1 min.
- Add 0.1N HCl for 10 min.
- Prepare the reverse transcription mixture on ice (important).
- 159 ul DEPC-H2O (Ambion)
- 20 ul 10x MMLV reaction buffer
- 2 ul 25 mM dNTP (Enzymatics)
- 2 ul 4 mM aminoallyl dUTP (Anaspec)
- 5 ul 100 uM /5Phos/TCTCGGGAACGCTGAAGANNNNNN
- 2 ul RNase Inhibitor (Enzymatics)
- 10 ul MMuLV reverse transcriptase (Enzymatics)
- 200ul Total Volume
- Add the enzyme cocktail to the fixed cells.
- Incubate at RT for 10 min, then transfer to 37°C for overnight.(Day 1)
- Aspirate.
- Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
- Block with 1M Tris pH 8.0 for 30 min.
- Add 10 ul Riboshredder (Epicentre) in T.E. for 1 hr at 37C.
- Rinse the entire well with ddH2O twice to remove traces of phosphate.
- Prepare the CircLigase reaction mixture.
- 60 ul ddH2O,
- 10 ul 10x CircLigase II reaction buffer,
- 5 ul 50 mM MnCl2,
- 20 ul 5 M Betaine,
- 5 ul CircLigase II enzyme
- 100 ul Total volume
- Place in a larger dish with wet towel and incubate at 60C for 2 hr.
- Aspirate the reaction mixture.
- Prepare the rolling circle amplification reaction mixtures on ice.
- 165 ul ddH2O,
- 20 ul 10x Phi29 buffer,
- 2 ul 25 mM dNTP,
- 2 ul 4 mM aminoallyl dUTP
- 1 ul 100 uM RCA primer (TCTTCAGCGTTCCCGA*G*A)
- 10 ul Phi29 DNA polymerase
- 200ul Total Volume
- Add to cells/tissue and incubate overnight at 30C.
- Wash with PBS once.
- Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
- Aspirate well and add 1M Tris pH 8.0 for 30 min.
- Store in T.E.
In Situ Whole Transcriptome Amplification From the Fixed Tissue Sections[edit]
- Wash with DEPC-PBS.
- Incubate with 0.01% Pepsin (Roche) in 0.1 HCl for 30 min.
- Prepare the reverse transcription mixture on ice.
- 159 ul DEPC-H2O (Ambion)
- 20 ul 10x MMLV reaction buffer
- 2 ul 25 mM dNTP (Enzymatics)
- 2 ul 4 mM aminoallyl dUTP (Anaspec)
- 5 ul 100 uM /5Phos/TCTCGGGAACGCTGAAGANNNNNN
- 2 ul RNase Inhibitor (Enzymatics)
- 10 ul MMLuV reverse transcriptase (Enzymatics)
- 200 ul Total volume
- Add the enzyme cocktail to the fixed cells.
- Incubate at RT for 10 min, then transfer to 37°C for overnight.(Day 1)
- Aspirate.
- Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
- Block with 1M Tris pH 8.0 for 30 min.
- Add 10 ul DNase-free RNases (Roche) in 200 ul 1x NEB#2 for 1 hr at 37C.
- Prepare the CircLigase reaction mixture.
- 60 ul ddH2O,
- 10 ul 10x CircLigase II reaction buffer,
- 5 ul 50 mM MnCl2,
- 20 ul 5 M Betaine,
- 5 ul CircLigase II enzyme
- 100 ul Total volume
- Cover with 2 ml mineral oil and incubate at 60°C overnight.(Day 2)
- Aspirate the layer of mineral oil and the reaction mixture.
- Prepare the rolling circle amplification reaction mixtures.
- 165 ul ddH2O,
- 20 ul 10x Phi29 buffer,
- 2 ul 25 mM dNTP,
- 2 ul 4 mM aminoallyl dUTP,
- 1 ul 100 uM RCA primer (TCTTCAGCGTTCCCGA*G*A),
- 10 ul Phi29 DNA polymerase
- 200ul Total Volume
- Add to cells/tissue and incubate overnight at 30C.(Day 3)
- Wash with PBS on
- Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
- Aspirate well and add 1M Tris pH 8.0 for 30 min.
- Store in T.E.