Matt:LabNotes/2014-4-9: Difference between revisions

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===Results===
===Results===
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Position'''
| align="center" style="background:#f0f0f0;"|'''Well'''
| align="center" style="background:#f0f0f0;"|'''Ampligase'''
| align="center" style="background:#f0f0f0;"|'''Digestion'''
| align="center" style="background:#f0f0f0;"|'''Rolonies'''
|-
| 5||F3||-||Exo III||203
|-
| 6||F4||-||Exo I/III||146
|-
| 7||F5||-||-||302
|-
| 8||F6||-||-||230
|-
| 9||F7||+||-||71
|-
| 10||F8||+||Exo I/III||0
|-
| 11||F9||+||Exo III||174
|-
| 12||F10||+||-||195
|}


===Conclusion===
===Conclusion===

Revision as of 18:21, 11 April 2014

Efficiency of Ampligase in situ Test

  • 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

  • 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

  • Wash with 2X SSC once
  • Add 50ul Exonuclease mix
  • Incubate @37C for 2 hrs

Exo I/III: F4 & F8

Component Volume
10X Exo III Buffer 10
Exo I 5
Exo III 5
H2O 80
Total 100

Exo III: F3 & F9

Component Volume
10X Exo III Buffer 10
Exo III 5
H2O 85
Total 100

Control: F5, F6, F7 & F10

Component Volume
10X Exo III Buffer 20
H2O 180
Total 200

Detect

  • 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

Position Well Ampligase Digestion Rolonies
5 F3 - Exo III 203
6 F4 - Exo I/III 146
7 F5 - - 302
8 F6 - - 230
9 F7 + - 71
10 F8 + Exo I/III 0
11 F9 + Exo III 174
12 F10 + - 195

Conclusion