Hosuk:LabNotes/2013-5-2: Difference between revisions

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====Reverse Transcription check====
===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=====
=====Result=====
Line 67: Line 66:




====CircLigase II reaction on the dish====
----
----
 
 
===CircLigase II reaction on the dish===
*based on Debugging plan ([[Hosuk:LabNotes/2013-5-1|05/01]])
*based on Debugging plan ([[Hosuk:LabNotes/2013-5-1|05/01]])




=====Result=====
*Reaction for 2.5 hr
*Stop reaction after 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
Line 82: Line 84:
**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
*Run TBU gel
 
 
=====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

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