Hosuk:LabNotes/2013-5-2: Difference between revisions
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|[[File:Composite_NoRTase_fig07-08.jpg|300px]] | |[[File:Composite_NoRTase_fig07-08.jpg|300px]] | ||
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*Reaction for 2.5 hr | |||
* | |||
*Aspirate and transfer solution from dish to tube | *Aspirate and transfer solution from dish to tube | ||
*Total volume collected | *Total volume collected | ||
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**6uL of mineral oil sample + 1uL of Exo I | **6uL of mineral oil sample + 1uL of Exo I | ||
**45min at 37C --> 15min at 80C | **45min at 37C --> 15min at 80C | ||
=====Result===== | |||
*Run TBU gel | *Run TBU gel | ||
====Rolony test with using pre-circulated DNA==== | |||
*based on Debugging plan ([[Hosuk:LabNotes/2013-5-1|05/01]]) | |||
*RCA reaction for 18hour | |||
*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 | |||
*Use Cy5-Adpater primer as usual | |||
=====Result===== |
Revision as of 23:43, 2 May 2013
Reverse Transcription check
- based on Debugging plan (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
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
- 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
- Run TBU gel
Rolony test with using pre-circulated DNA
- based on Debugging plan (05/01)
- RCA reaction for 18hour
- 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
- Use Cy5-Adpater primer as usual