Hosuk:LabNotes/2013-11-19

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Revision as of 05:54, 19 November 2013 by >Hosuki78
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Dye labeled Probe Annealing Test – with Cy3-1st Rolony

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


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


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


Result

  • 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
    • 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 %

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


1st Rolonies along each step

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