Revision as of 00:26, 12 April 2014 by >Mzcai
Efficiency of Ampligase in situ Test[edit]
- General idea: Will use Exo I & Exo III OR Exo III only to digest non-ligased padlock probes, then use dcProbe2RevComp to hybridize to padlock probe and detect
- Exo III only because it's possible Exo I will degrade all rolonies, leaving the padlock probe (even after ligation) nothing to attach to so it'll be washed away and not detected
- Ligation with Ampligase should protect the padlock probes from digestion, hence comparing wells +Exo with those -Exo will show what percentage of padlock probes are ligated
Hybridize 2uM ppMALAT1[edit]
- Add 50ul 2uM ppMALAT1 for 21.5hr @45C (7:00pm-4:30pm ) in 1X Ampligase buffer +/- 0.5U/ul Ampligase
F3-F6: -Ampligase
Component
|
Volume
|
10X Ampligase Buffer |
20
|
200uM ppMALAT1 |
2
|
H2O |
178
|
Total |
200
|
F7-F10: + Ampligase
Component
|
Volume
|
10X Ampligase Buffer |
20
|
200uM ppMALAT1 |
2
|
5 U/ul Ampligase |
20
|
H2O |
158
|
Total |
200
|
Digestion[edit]
- Wash with 2X SSC once
- Add 50ul Exonuclease mix
- Incubate @37C for 2 hrs
Exo I/III: F4 & F8[edit]
Component
|
Volume
|
10X Exo III Buffer |
10
|
Exo I |
5
|
Exo III |
5
|
H2O |
80
|
Total |
100
|
Exo III: F3 & F9[edit]
Component
|
Volume
|
10X Exo III Buffer |
10
|
Exo III |
5
|
H2O |
85
|
Total |
100
|
Control: F5, F6, F7 & F10[edit]
Component
|
Volume
|
10X Exo III Buffer |
20
|
H2O |
180
|
Total |
200
|
- Wash with 2X SSC twice
- Add 50ul 1uM dcProbe2RevComp-Cy3 (ATTO 488 dye)
- Pre-heated to 75C then snap-cooled before adding
- Wash with 2X SSC twice
- Images saved in 4-10-2014
Results[edit]
Position
|
Well
|
Ampligase
|
Digestion
|
Hybridized Padlock Probes
|
Rolonies
|
%
|
5 |
F3 |
- |
Exo III |
203 |
15,805 |
1.28
|
6 |
F4 |
- |
Exo I/III |
146 |
7,253 |
2.01
|
7 |
F5 |
- |
- |
302 |
7,754 |
3.89
|
8 |
F6 |
- |
- |
230 |
6,210 |
3.70
|
9 |
F7 |
+ |
- |
71 |
14,548 |
0.49
|
10 |
F8 |
+ |
Exo I/III |
0 |
13,323 |
0.00
|
11 |
F9 |
+ |
Exo III |
174 |
27,904 |
0.62
|
12 |
F10 |
+ |
- |
195 |
22,660 |
0.86
|
- Using bkg_mult = 3 because a lot of features weren't detected as "polonies"
- Also summed the intensity values of each pixel in an image since it should correlate more with number of tagged padlock probes rather than # of rolonies tagged by padlock probes
Position
|
Well
|
Ampligase
|
Digestion
|
Hybridized Padlock Probes
|
Rolonies
|
%
|
Intensity*Area
|
5 |
F3 |
- |
Exo III |
251 |
15,805 |
1.59 |
34,553,526
|
6 |
F4 |
- |
Exo I/III |
191 |
7,253 |
2.63 |
24,825,840
|
7 |
F5 |
- |
- |
311 |
7,754 |
4.01 |
53,719,236
|
8 |
F6 |
- |
- |
310 |
6,210 |
4.99 |
47,879,730
|
9 |
F7 |
+ |
- |
107 |
14,548 |
0.74 |
31,531,795
|
10 |
F8 |
+ |
Exo I/III |
0 |
13,323 |
0.00 |
19,118,820
|
11 |
F9 |
+ |
Exo III |
222 |
27,904 |
0.80 |
42,094,484
|
12 |
F10 |
+ |
- |
245 |
22,660 |
1.08 |
49,782,492
|
File:20140410 EfficiencyAmpligaseinsituTest.JPG
Conclusion[edit]
- Exonuclease digestion did not seem complete
- Possibly adding 2uM of ppMALAT1 is too much for Exo I/III to handle in 2hrs? but doubt it
- Use 10nM ppMALAT1 next time to match the Exo I/III test
- Because -Ampligase wells with +Exo still detected signal, cannot draw any conclusions about +Ampligase wells
- The average % of detected rolonies of +Ampligase wells is less than average of -Ampligase wells:
- Does Ampligase decrease the efficiency % of hybridization?
- Or is it because the +Ampligase wells had more rolonies to start with?
- If that were true, it would imply ppMALAT1 is limiting reagent, which is unlikely given 2uM
- Try adding 50% glycerol in -Ampligase wells next time to better mimic +Ampligase wells