Matt:LabNotes/2014-4-15: Difference between revisions

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(Created page with "==Efficiency of Ampligase in situ Test: Second Try== * Previously results were not good enough to draw clear conclusion *Samples are wells A5, D2,...")
 
>Mzcai
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===Hybridize 10nM ppMALAT1===
===Hybridize 10nM ppMALAT1===
*Add 100ul 10nM ppMALAT1 for 21.5hr @45C in 1X Ampligase buffer +/- 0.5U/ul Ampligase
*Add 100ul 10nM ppMALAT1 for 24hr @45C in 1X Ampligase buffer +/- 0.5U/ul Ampligase


A5&D2: -Ampligase
A5&D2: -Ampligase
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| Total||300
| Total||300
|}
|}
<!--
 
===Digestion===
===Digestion===
*Wash with 2X SSC once
*Wash with 2X SSC once
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*Incubate @37C for 2 hrs
*Incubate @37C for 2 hrs


====Exo I/III: F4 & F8====
====Exo I/III: D3====
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
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|}
|}


====Exo III: F3 & F9====
====Exo III: D2&D4====
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| 10X Exo III Buffer||10
| 10X Exo III Buffer||20
|-
|-
| Exo III||5
| Exo III||10
|-
|-
| H2O||85
| H2O||170
|-
|-
| Total||100
| Total||200
|}
|}


====Control: F5, F6, F7 & F10====
====Control: A5&D5====
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
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**Pre-heated to 75C then snap-cooled before adding
**Pre-heated to 75C then snap-cooled before adding
*Wash with 2X SSC twice
*Wash with 2X SSC twice
-->

Revision as of 21:02, 16 April 2014

Efficiency of Ampligase in situ Test: Second Try

  • Previously results were not good enough to draw clear conclusion
  • 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 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 10nM ppMALAT1

  • Add 100ul 10nM ppMALAT1 for 24hr @45C in 1X Ampligase buffer +/- 0.5U/ul Ampligase

A5&D2: -Ampligase

Component Volume
10X Ampligase Buffer 20
1uM ppMALAT1 2
H2O 178
Total 200

D3,D4,&D5: + Ampligase

Component Volume
10X Ampligase Buffer 30
1uM ppMALAT1 3
5 U/ul Ampligase 30
H2O 237
Total 300

Digestion

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

Exo I/III: D3

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

Exo III: D2&D4

Component Volume
10X Exo III Buffer 20
Exo III 10
H2O 170
Total 200

Control: A5&D5

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