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===Reverse Transcription check=== *based on Debugging plan ([[Hosuk:LabNotes/2013-5-1|05/01]]) =====Result===== *RT process seemed work. *+RTase sample showed very strong Cy5 signal which is supposed cDNA *comparing to -RTase result, the bright singal over the cell seemed they were cDNAs *-RTase sample has very low intensity of signals in the cell, and *not many spots were observed, which spots were outside of cells. *Most of signal came from cell structure. *The picture of -RTase is the position where the most number of spots were observed in all over the area. *In the +RTase sample, similar number or density of spots were observed in all over the area. *I don't know what are the large spots. Are they also cDNAs? *'''+RTase''' {| {{table}} | S2:+RTase, Brighfield, 20x || | S2:+RTase, Cy5, 20x || | S2:+RTase, Merged || |- |([[Media:RTCheck_+RTase_fig08_20xobj_pos04_BF_exp0.2-EM00.tif|Original]]) || |([[Media:RTCheck_+RTase_fig07_20xobj_pos04_Cy5_exp0.2-EM40.tif|Original]]) || | |- |[[File:RTCheck_+RTase_fig08_20xobj_pos04_BF_exp0.2-EM00.jpg|300px]] || |[[File:RTCheck_+RTase_fig07_20xobj_pos04_Cy5_exp0.2-EM40.jpg|300px]] || |[[File:Composite_+RTase_fig07-08.jpg|300px]] |} *'''-RTase (No RTase)''' {| {{table}} | S3:-RTase, Brighfield, 20x || | S3:-RTase, Cy5, 20x || | S3:-RTase, Merged || |- |([[Media:RTCheckNoRTase_fig08_20xobj_pos05_BF_exp0.2-EM00.tif|Original]]) || |([[Media:RTCheckNoRTase_fig07_20xobj_pos04_Cy5_exp0.2-EM40.tif|Original]]) || | |- |[[File:RTCheckNoRTase_fig08_20xobj_pos05_BF_exp0.2-EM00.jpg|300px]] || |[[File:RTCheckNoRTase_fig07_20xobj_pos04_Cy5_exp0.2-EM40.jpg|300px]] || |[[File:Composite_NoRTase_fig07-08.jpg|300px]] |} ---- ---- ===CircLigase II reaction on the dish=== *based on Debugging plan ([[Hosuk:LabNotes/2013-5-1|05/01]]) *Reaction for 2.5 hr *Aspirate and transfer solution from dish to tube *Total volume collected **8~9uL from the sample at wet towel **about 50uL from the sample at mineral oil, but this volume include oil. After spinning the actual reaction solution seemed 8~10uL *Run Exo I reaction **9uL of pre-circulated DNA + 1uL of Exo I **6uL of wet towel sample + 1uL of Exo I **6uL of mineral oil sample + 1uL of Exo I **45min at 37C --> 15min at 80C =====Result===== *CircLigase II reaction worked on a dish well *Wet towel method looked better efficiency than oil cover method did. *[[File:GelTable_CircLigaseIIonDish_2.jpg|550px]] *[[File:CircOnDish_2_edit.jpg|350px]] ---- ---- ===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]] |} ---- ---- ===Intermediate thinking=== *Comparison of each step **Each picture is from different samples. [[File:Compare_each_step_050213_2.jpg|900px]] *Pre-circularized DNA didn't seem bingin well in cell structure. (Why? due to BS(PEG)9 step?) *Need to think more what's the major problem, or bottleneck
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