Hosuk:LabNotes/2013-11-19: Difference between revisions

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====Procedure====
====Procedure====
*2048 x 2-48, 600Hz Image quality is not different from 400Hz.
*20x obj, 2048 x 2048 resolution, 16bit,
*Scan Freq. 600Hz --> Image quality is not different from 400Hz.
*Sample : MatTek Dish, made at 07/09
*Sample : MatTek Dish, made at 07/09
*Each stripping, and annealing : 15min incubation @ RT
*Each stripping, and annealing : 15min incubation @ RT
*Each time the concentration of the dye-labeled probe is 2uM,
*The volume added is 100uL.




*Process Steps
*Process Steps
**Step 0 : Strip
*#Step 0 : Strip
**Step 1 : Cy5 in 2x SSC
*#Step 1 : Cy5 in 2x SSC
**Step 2 : Strip
*#Step 2 : Strip
**Step 3 : Cy5 in 70% Formamide + 2x SSC
*#Step 3 : Cy5 in 70% Formamide + 2x SSC
**Step 4 : Strip
*#Step 4 : Strip
**Step 5 : Cy5 in 10% Formamide + 2x SSC
*#Step 5 : Cy5 in 10% Formamide + 2x SSC
**Step 6 : Strip
*#Step 6 : Strip
**Step 7 : Cy5 in 30 % Formamide + 2x SSC
*#Step 7 : Cy5 in 30 % Formamide + 2x SSC
**Step 8 : Strip
*#Step 8 : Strip
**Step 9 : Cy5 in 50% Formamide + 2x SSC
*#Step 9 : Cy5 in 50% Formamide + 2x SSC
**Step 10 : Strip
*#Step 10 : Strip
**Step 11 : Cy5 in 70% Formamide + 2x SSC
*#Step 11 : Cy5 in 70% Formamide + 2x SSC




Line 34: Line 37:




*1st Rolony Count
=====1st Rolony Count=====
**Before Step 0 (At the beginning Cy3 probe were annealed) --> 5932
*Before Step 0 (At the beginning Cy3 probe were annealed) --> 5932
**Step 1  : Cy5 in 2x SSC --> 5469
*Step 1  : Cy5 in 2x SSC --> 5469
**Step 3  : Cy5 in 70% Formamide + 2x SSC --> 543
*Step 3  : Cy5 in 70% Formamide + 2x SSC --> 543
**Step 5  : Cy5 in 10% Formamide + 2x SSC --> 5413
*Step 5  : Cy5 in 10% Formamide + 2x SSC --> 5413
**Step 7  : Cy5 in  30 % Formamide + 2x SSC --> 5446
*Step 7  : Cy5 in  30 % Formamide + 2x SSC --> 5446
**Step 9  : Cy5 in 50% Formamide + 2x SSC --> 4793
*Step 9  : Cy5 in 50% Formamide + 2x SSC --> 4793
**Step 11 : Cy5 in 70% Formamide + 2x SSC --> 421
*Step 11 : Cy5 in 70% Formamide + 2x SSC --> 421
 




Line 62: Line 64:




[[File:Chart_DyeProbeTest_Cy5-1stRolony_Step1-3-5-7-9-11.png|600px]]
=====Number of 1st Rolony vs. Formamide %=====
[[File:Chart_DyeProbeTest_Cy5-1stRolony_Step1-3-5-7-9-11.png|850px]]
 
 
=====1st Rolonies along each step=====
[[File:DyeProbeTest_Cy5-1stRolony_Step1-3-5-7-9-11_label.png|850px]]
*'''Raw images'''
**[[Media:DyeProbeTest_MatTek20130709_1stRolony_Cy3_2k_600Hz_ch00_MIP.tif|Before Step0 (Cy3, ]][[Media:DyeProbeTest_MatTek20130709_1stRolony_Cy3_2k_600Hz_ch01_MIP.tif|BF)]],
**[[Media:DyeProbeTest.lif_MatTek20130709_Step1_Cy5_ch00_MIP.tif|Step1 (Cy5]], [[Media:DyeProbeTest.lif_MatTek20130709_Step1_Cy5_ch01_MIP.tif|BF)]], [[Media:DyeProbeTest.lif_MatTek20130709_Step3_Cy5-Formamide70_Cy5_ch00_MIP.tif|Step3]], [[Media:DyeProbeTest.lif_MatTek20130709_Step5_Cy5-Formamide10_Cy5_ch00_MIP.tif|Step5]], [[Media:DyeProbeTest.lif_MatTek20130709_Step7_Cy5-Formamide30_Cy5_ch00_MIP.tif|Step7]], [[Media:DyeProbeTest.lif_MatTek20130709_Step9_Cy5-Formamide50_Cy5_ch00_MIP.tif|Step9 (Cy5, ]][[Media:DyeProbeTest.lif_MatTek20130709_Step9_Cy5-Formamide50_Cy5_ch01_MIP.tif|BF)]], [[Media:DyeProbeTest.lif_MatTek20130709_Step11_Cy5-Formamide70_Cy5_ch00_MIP.tif|Step11]]
*'''Stage was moveed a little from Step9 by mistake, so the location was re-configured.'''
 
 
 
 
===Dye labeled Probe Annealing Test – with dcProbe3-Cy3 : Non-specific binding===
*Figure out lower limit by Formamide concentration : to minimize non-specific binding
*dcProbe3-Cy3 does not have target in rolony, so we can see how many spots due to the non-specific binding.
*Done @ 11/19
 
 
====Procedure====
*20x obj, 2048 x 2048 resolution, 16bit,
*Scan Freq. 600Hz --> Image quality is not different from 400Hz.
*Sample : MatTek Dish, made at 07/09
*Each stripping, and annealing : 15min incubation @ RT
*Each time the concentration of the dye-labeled probe is 1uM,
*The volume added is 100uL.
 
 
*Process Steps
Process Step
Step 0 : Strip
Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC  160
Step 2 : Strip  ~34
Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC  67
Step 4 : Strip
Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC  66
 
*#Step 0 : Strip
*#Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC
*#Step 2 : Strip
*#Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC
*#Step 4 : Strip
*#Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC
 
 
====Result====
*30% seemed optimal concentration for less non-specific binding as well as for target-specific binding..
*Need to get 3 data point to confirm those results (for the future when I set up an automation system.
 
 
=====Spot Count=====
 
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Formamide (%)'''
| align="center" style="background:#f0f0f0;"|'''Spot Count'''
|-
| align="center" | Strip || align="center" | 34
|-
| align="center" | 0 || align="center" | 160
|-
| align="center" | 30 || align="center" | 67
|-
| align="center" | 40 || align="center" | 66
|}
 
 
[[File:Chart_DyeProbeTest_dcProbe3_Step1-2-3-5.png|800px]]
 
 
*'''Raw images'''
**[[Media:DyeProbeTest.lif_MatTek20130709_Step1_dcProbe3_Cy3_ch00_MIP.tif|Step1 (Cy3, ]][[Media:DyeProbeTest.lif_MatTek20130709_Step1_dcProbe3_Cy3_ch01_MIP.tif|BF)]], [[Media:DyeProbeTest.lif_MatTek20130709_Step2_Strip_Cy3_ch00_MIP.tif|Step2]], [[Media:DyeProbeTest.lif_MatTek20130709_Step3_dcProbe3_Formamide30_Cy3_ch00_MIP.tif|Step3]], [[Media:DyeProbeTest.lif_MatTek20130709_Step4_Strip_Cy3_ch00_MIP.tif|Step4]], [[Media:DyeProbeTest.lif_MatTek20130709_Step5_dcProbe3_Formamide40_Cy3_ch00_MIP.tif|Step5]]

Latest revision as of 05:37, 20 November 2013


Dye labeled Probe Annealing Test – with Cy3-1st Rolony[edit]

  • Figure out upper limit by Formamide concentration
  • Done @ 11/18


Procedure[edit]

  • 20x obj, 2048 x 2048 resolution, 16bit,
  • Scan Freq. 600Hz --> Image quality is not different from 400Hz.
  • Sample : MatTek Dish, made at 07/09
  • Each stripping, and annealing : 15min incubation @ RT
  • Each time the concentration of the dye-labeled probe is 2uM,
  • The volume added is 100uL.


  • Process Steps
    1. Step 0 : Strip
    2. Step 1 : Cy5 in 2x SSC
    3. Step 2 : Strip
    4. Step 3 : Cy5 in 70% Formamide + 2x SSC
    5. Step 4 : Strip
    6. Step 5 : Cy5 in 10% Formamide + 2x SSC
    7. Step 6 : Strip
    8. Step 7 : Cy5 in 30 % Formamide + 2x SSC
    9. Step 8 : Strip
    10. Step 9 : Cy5 in 50% Formamide + 2x SSC
    11. Step 10 : Strip
    12. Step 11 : Cy5 in 70% Formamide + 2x SSC


Result[edit]

  • 0~30% Formamide concentration showed similar number of rolony, but the number decreased at 70%.
  • Need to try 20%, 30%, 40%, twice at each time in order to confirm 30% is maximum condition!


1st Rolony Count[edit]
  • Before Step 0 (At the beginning Cy3 probe were annealed) --> 5932
  • Step 1  : Cy5 in 2x SSC --> 5469
  • Step 3  : Cy5 in 70% Formamide + 2x SSC --> 543
  • Step 5  : Cy5 in 10% Formamide + 2x SSC --> 5413
  • Step 7  : Cy5 in 30 % Formamide + 2x SSC --> 5446
  • Step 9  : Cy5 in 50% Formamide + 2x SSC --> 4793
  • Step 11 : Cy5 in 70% Formamide + 2x SSC --> 421


Formamide (%) 1st Rolony Count
0 5469
10 5413
30 5446
50 4793
70 482
  • 0% : average from 2 results


Number of 1st Rolony vs. Formamide %[edit]

File:Chart DyeProbeTest Cy5-1stRolony Step1-3-5-7-9-11.png


1st Rolonies along each step[edit]

File:DyeProbeTest Cy5-1stRolony Step1-3-5-7-9-11 label.png



Dye labeled Probe Annealing Test – with dcProbe3-Cy3 : Non-specific binding[edit]

  • Figure out lower limit by Formamide concentration : to minimize non-specific binding
  • dcProbe3-Cy3 does not have target in rolony, so we can see how many spots due to the non-specific binding.
  • Done @ 11/19


Procedure[edit]

  • 20x obj, 2048 x 2048 resolution, 16bit,
  • Scan Freq. 600Hz --> Image quality is not different from 400Hz.
  • Sample : MatTek Dish, made at 07/09
  • Each stripping, and annealing : 15min incubation @ RT
  • Each time the concentration of the dye-labeled probe is 1uM,
  • The volume added is 100uL.


  • Process Steps

Process Step Step 0 : Strip Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC  160 Step 2 : Strip  ~34 Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC  67 Step 4 : Strip Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC  66

    1. Step 0 : Strip
    2. Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC
    3. Step 2 : Strip
    4. Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC
    5. Step 4 : Strip
    6. Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC


Result[edit]

  • 30% seemed optimal concentration for less non-specific binding as well as for target-specific binding..
  • Need to get 3 data point to confirm those results (for the future when I set up an automation system.


Spot Count[edit]
Formamide (%) Spot Count
Strip 34
0 160
30 67
40 66


File:Chart DyeProbeTest dcProbe3 Step1-2-3-5.png