Hosuk:LabNotes/2013-5-1: Difference between revisions

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*Prepare two samples of fixed cell, and make one dish as a control without RTase
*Prepare two samples of fixed cell, and make one dish as a control without RTase
*Detail procedure
*Detail procedure
**Add 0.1N HCl  for 10 min.
**Wash with PBS twice
**Prepare the reverse transcription mixture on ice (important).
{| {{table}}
| 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.





Revision as of 21:22, 2 May 2013

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
    1. Fix cells and permeabilize using 70% ethanol as before
    2. Mix 5 ul fluorescent primer (Cy3-polydT(50 mer)) in 200 ul 2xSSC/10% dextran sulfate/10% formamide
    3. Add to cells and incubate at 37C for 1 hour
    4. Wash using 2xSSC/10% formamide for 15 min each twice
    5. 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
    1. Add the enzyme cocktail to the fixed cells.
    2. Incubate at RT for 10 min, then transfer to 37°C for overnight.
    3. Aspirate.
    4. Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
    5. Wash with PBS once
    6. Block with 1M Tris pH 8.0 for 30 min.
    7. Wash with PBS once
    8. Add 5 ul Riboshredder (Epicentre)+ 5ul RNase H(Enzymatics) in 190ul RNase H buffer(Enzymatics) for 1 hr at 37C.
    9. 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'