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===Rolony test with using pre-circulated DNA=== *based on Debugging plan ([[Hosuk:LabNotes/2013-5-1|05/01]]) *RCA reaction for 18hour *Add BS(PEG)9 (20uL BS(PEG)9 + 980uL PBS) **S4 : Aspirate, then wash with PBS once, then add BS(PEG)9 **S5 : Aspirate, then add BS(PEG)9 **S6 : Aspirate, then wash with PBS once, then add BS(PEG)9 *Block with Tris pH 8.0 : Aspirate, then wash with PBS once, then add Tris pH 8.0 *Wash with 2x SSC once *Cy3-Adpater primer as usual =====Result===== *'''S4 : RCA with Linear DNA''' *No RCA product was observed. *No RCA is done with linead DNA, same as the result at [[Hosuk:LabNotes/2013-4-23|04/23]]. {| {{table}} | Bright field, 20x || | Cy3, 20x |- |([[Media:CircTest_S4-LinearDNA_fig04_20x_pos02_BF.tif|Original]]) || |([[Media:CircTest_S4-LinearDNA_fig03_20x_pos02_Cy3.tif|Original]]) |- |[[File:CircTest_S4-LinearDNA_fig04_20x_pos02_BF.jpg|300px]] || |[[File:CircTest_S4-LinearDNA_fig03_20x_pos02_Cy3.jpg|300px]] |} ---- *'''S5 : RCA with pre-circularized DNA, add BS(PEG)9''' *RCA product was observed, but also there were a lot of linear-like patterns. *Some area has less 'linear pattern', but most area has this linear pattern. *And the signal was not as much as that from reverse transcription result. *Washing well is very important for clear signal, *But still the question is how to get more rolonies within cells... *'''S5, observation position 1''' {| {{table}} | Bright field, 20x || | Cy3, 20x || | Merged || |- |([[Media:CircTest_S5-CircDNA-NoWash_fig02_20x_pos01_BF.tif|Original]]) || |([[Media:CircTest_S5-CircDNA-NoWash_fig01_20x_pos01_Cy3.tif|Original]]) || | |- |[[File:CircTest_S5-CircDNA-NoWash_fig02_20x_pos01_BF.jpg|300px]] || |[[File:CircTest_S5-CircDNA-NoWash_fig01_20x_pos01_Cy3.jpg|300px]] || |[[File:Composite_CircRCA_S5_fig01-02.jpg|300px]] |} *'''S5, observation position 2''' {| {{table}} | Bright field, 20x || | Cy3, 20x || | Merged || |- |([[Media:CircTest_S5-CircDNA-NoWash_fig04_20x_pos02_BF.tif|Original]]) || |([[Media:CircTest_S5-CircDNA-NoWash_fig03_20x_pos02_Cy3.tif|Original]]) || | |- |[[File:CircTest_S5-CircDNA-NoWash_fig04_20x_pos02_BF.jpg|300px]] || |[[File:CircTest_S5-CircDNA-NoWash_fig03_20x_pos02_Cy3.jpg|300px]] || |[[File:Composite_CircRCA_S5_fig03-04.jpg|300px]] |} ---- *'''S6 : RCA with pre-circularized DNA, add BS(PEG)9''' *RCA product was observed, and there were less linear-like patterns than S5's. *However the signal was still not as much as that from reverse transcription result. *Again, washing step need to be optimized more. *And also how to get more rolonies within cells... *'''S6, observation position 2''' {| {{table}} | Bright field, 20x || | Cy3, 20x || | Merged || |- |([[Media:CircTest_S6-CircDNA-Wash_fig04_20x_pos02_BF.tif|Original]]) || |([[Media:CircTest_S6-CircDNA-Wash_fig03_20x_pos02_Cy3.tif|Original]]) || | |- |[[File:CircTest_S6-CircDNA-Wash_fig04_20x_pos02_BF.jpg|300px]] || |[[File:CircTest_S6-CircDNA-Wash_fig03_20x_pos02_Cy3.jpg|300px]] || |[[File:Composite_CircRCA_S6_fig03-04.jpg|300px]] |} *'''S6, observation position 5''' {| {{table}} | Bright field, 20x || | Cy3, 20x || | Merged || |- |([[Media:CircTest_S6-CircDNA-Wash_fig10_20x_pos05_BF.tif|Original]]) || |([[Media:CircTest_S6-CircDNA-Wash_fig09_20x_pos05_Cy3.tif|Original]]) || | |- |[[File:CircTest_S6-CircDNA-Wash_fig10_20x_pos05_BF.jpg|300px]] || |[[File:CircTest_S6-CircDNA-Wash_fig09_20x_pos05_Cy3.jpg|300px]] || |[[File:Composite_CircRCA_S6_fig09-10.jpg|300px]] |} ---- ----
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