Hosuk:LabNotes/2013-5-1: Difference between revisions
Jump to navigation
Jump to search
>Hosuki78 No edit summary |
>Hosuki78 No edit summary |
||
Line 9: | Line 9: | ||
*#Add to cells and incubate at 37C for 1 hour | *#Add to cells and incubate at 37C for 1 hour | ||
*#Wash using 2xSSC/10% formamide for 15 min each twice | *#Wash using 2xSSC/10% formamide for 15 min each twice | ||
*#Aspirate and add 2x SSC | *#Aspirate and add 2x SSC and then image | ||
===== | =====2. 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 | |||
=====3. 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]]) | |||
*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 | |||
=====4. Rolony test with using pre-circulated DNA===== | |||
*This test was done at [[Hosuk:LabNotes/2013-4-24|04/24]], but try one more time. | |||
*''''Since washing step'''' |
Revision as of 21:13, 2 May 2013
Debugging plan (re-organized)
- Re-planning from the previous debugging plan (04/01)
1. 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
2. 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
3. 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
4. Rolony test with using pre-circulated DNA
- This test was done at 04/24, but try one more time.
- 'Since washing step'