Hosuk:LabNotes/2013-11-19

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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