Hosuk:LabNotes/2013-5-1
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Debugging plan (re-organized)
- Re-planning from the previous debugging plan (04/01)
mRNA population check after fixing
- mRNA were observed from the previous result(03/17), but just do again with the new samples fixed at 05/01
- Detail procedure
- Fix cells and permeabilize using 70% ethanol as before
- Mix 5 ul fluorescent primer (Cy3-polydT(50 mer)) in 200 ul 2xSSC/10% dextran sulfate/10% formamide
- Add to cells and incubate at 37C for 1 hour
- Wash using 2xSSC/10% formamide for 15 min each twice
- Aspirate and add 2x SSC and then image
Reverse Transcript check
- Test if RT process works well
- Prepare two samples of fixed cell, and make one dish as a control without RTase
- Detail procedure
- Add 0.1N HCl for 10 min.
- Wash with PBS twice
- Prepare the reverse transcription mixture on ice (important).
Reagent | Volume | |
DEPC-H2O (Ambion) | 159uL | |
10x MMLV reaction buffer | 20uL | |
25 mM dNTP (Enzymatics) | 2uL | |
4 mM aminoallyl dUTP (Anaspec) | 2uL | |
100 uM /5Phos/TCTCGGGAACGCTGAAGANNNNNN | 5uL | |
RNase Inhibitor (Enzymatics) | 2uL | |
MMuLV reverse transcriptase (Enzymatics) | 10uL |
- Add the enzyme cocktail to the fixed cells.
- Incubate at RT for 10 min, then transfer to 37°C for overnight.
- Aspirate.
- Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
- Wash with PBS once
- Block with 1M Tris pH 8.0 for 30 min.
- Wash with PBS once
- Add 5 ul Riboshredder (Epicentre)+ 5ul RNase H(Enzymatics) in 190ul RNase H buffer(Enzymatics) for 1 hr at 37C.
- Rinse the entire well with ddH2O twice to remove traces of phosphate.
CircLigase II reaction on the dish incubating in the oven
- CircLigase II reaction in a tube was confirmed (04/22)
- Test if CircLigase II reaction works well on the dish, and using 60C oven
- Also, in Jay's protocol, minetal oil covering is used for preventing evaporation
- Thus I need to compare two same CircLigase II mix on the glass bottom MatTek dish, but one dish is on the wet towel, another one is covered by mineral oil.
- And then run a gel with two product by extract volume
- Detail procedure
Rolony test with using pre-circulated DNA
- This test was done at 04/24, but try one more time.
- 'Since washing step'