Hosuk:LabNotes/2013-5-2: Difference between revisions
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=== | ===Reverse Transcription check=== | ||
*based on Debugging plan ([[Hosuk:LabNotes/2013-5-1|05/01]]) | *based on Debugging plan ([[Hosuk:LabNotes/2013-5-1|05/01]]) | ||
=====Result===== | |||
*RT process seemed work. | *RT process seemed work. | ||
Line 10: | Line 10: | ||
*-RTase sample has very low intensity of signals in the cell, and | *-RTase sample has very low intensity of signals in the cell, and | ||
*not many spots were observed, which spots were outside of cells. | *not many spots were observed, which spots were outside of cells. | ||
*Most of signal came from cell structure. | *Most of signal came from cell structure. | ||
Line 18: | Line 16: | ||
*In the +RTase sample, similar number or density 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? | *I don't know what are the large spots. Are they also cDNAs? | ||
Line 69: | Line 66: | ||
====Result | ---- | ||
---- | |||
===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]]) | *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 |
Latest revision as of 17:54, 6 May 2013
Reverse Transcription check[edit]
- based on Debugging plan (05/01)
Result[edit]
- 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
S2:+RTase, Brighfield, 20x | S2:+RTase, Cy5, 20x | S2:+RTase, Merged | |||
(Original) | (Original) | ||||
File:RTCheck +RTase fig08 20xobj pos04 BF exp0.2-EM00.jpg | File:RTCheck +RTase fig07 20xobj pos04 Cy5 exp0.2-EM40.jpg | File:Composite +RTase fig07-08.jpg |
- -RTase (No RTase)
S3:-RTase, Brighfield, 20x | S3:-RTase, Cy5, 20x | S3:-RTase, Merged | |||
(Original) | (Original) | ||||
File:RTCheckNoRTase fig08 20xobj pos05 BF exp0.2-EM00.jpg | File:RTCheckNoRTase fig07 20xobj pos04 Cy5 exp0.2-EM40.jpg | File:Composite NoRTase fig07-08.jpg |
CircLigase II reaction on the dish[edit]
- based on Debugging plan (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[edit]
- CircLigase II reaction worked on a dish well
- Wet towel method looked better efficiency than oil cover method did.
Rolony test with using pre-circulated DNA[edit]
- based on Debugging plan (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[edit]
- S4 : RCA with Linear DNA
- No RCA product was observed.
- No RCA is done with linead DNA, same as the result at 04/23.
Bright field, 20x | Cy3, 20x | |
(Original) | (Original) | |
File:CircTest S4-LinearDNA fig04 20x pos02 BF.jpg | File:CircTest S4-LinearDNA fig03 20x pos02 Cy3.jpg |
- 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
Bright field, 20x | Cy3, 20x | Merged | |||
(Original) | (Original) | ||||
File:CircTest S5-CircDNA-NoWash fig02 20x pos01 BF.jpg | File:CircTest S5-CircDNA-NoWash fig01 20x pos01 Cy3.jpg | File:Composite CircRCA S5 fig01-02.jpg |
- S5, observation position 2
Bright field, 20x | Cy3, 20x | Merged | |||
(Original) | (Original) | ||||
File:CircTest S5-CircDNA-NoWash fig04 20x pos02 BF.jpg | File:CircTest S5-CircDNA-NoWash fig03 20x pos02 Cy3.jpg | File:Composite CircRCA S5 fig03-04.jpg |
- 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
Bright field, 20x | Cy3, 20x | Merged | |||
(Original) | (Original) | ||||
File:CircTest S6-CircDNA-Wash fig04 20x pos02 BF.jpg | File:CircTest S6-CircDNA-Wash fig03 20x pos02 Cy3.jpg | File:Composite CircRCA S6 fig03-04.jpg |
- S6, observation position 5
Bright field, 20x | Cy3, 20x | Merged | |||
(Original) | (Original) | ||||
File:CircTest S6-CircDNA-Wash fig10 20x pos05 BF.jpg | File:CircTest S6-CircDNA-Wash fig09 20x pos05 Cy3.jpg | File:Composite CircRCA S6 fig09-10.jpg |
Intermediate thinking[edit]
- Comparison of each step
- Each picture is from different samples.
File:Compare each step 050213 2.jpg
- 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