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
- 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[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
- Raw images
- 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[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
- 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[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