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

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*Re-planning from the previous debugging plan ([[Hosuk:LabNotes/2013-4-1|04/01]])
*Re-planning from the previous debugging plan ([[Hosuk:LabNotes/2013-4-1|04/01]])


=====1. mRNA population check after fixing=====
=====mRNA population check after fixing=====
*mRNA were observed from the previous result([[Hosuk:LabNotes/2013-3-17|03/17]]), but just do again with the new samples fixed at 05/01
*mRNA were observed from the previous result([[Hosuk:LabNotes/2013-3-17|03/17]]), but just do again with the new samples fixed at 05/01
*Detail procedure
*Detail procedure
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=====2. Reverse Transcript check=====
=====Reverse Transcript check=====
*Test if RT process works well
*Test if RT process works well
*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
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=====3. CircLigase II reaction on the dish incubating in the oven=====
=====CircLigase II reaction on the dish incubating in the oven=====
*CircLigase II reaction in a tube was confirmed ([[Hosuk:LabNotes/2013-4-22|04/22]])
*CircLigase II reaction in a tube was confirmed ([[Hosuk:LabNotes/2013-4-22|04/22]])
*Test if CircLigase II reaction works well on the dish, and using 60C oven
*Test if CircLigase II reaction works well on the dish, and using 60C oven
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=====4. Rolony test with using pre-circulated DNA=====
=====Rolony test with using pre-circulated DNA=====
*This test was done at [[Hosuk:LabNotes/2013-4-24|04/24]], but try one more time.
*This test was done at [[Hosuk:LabNotes/2013-4-24|04/24]], but try one more time.
*''''Since washing step''''
*''''Since washing step''''

Revision as of 21:13, 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


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'