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]]) | ||
===== | =====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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===== | =====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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===== | =====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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===== | =====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
- 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
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'