Matt:LabNotes/2014-5-16: Difference between revisions

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(Created page with "==Detecting ppMALAT1 Hybridization== *Use sample from Matt:LabNotes/2014-5-7 **A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2-RevComp in one step **D3 & D4: Hybridi...")
 
>Mzcai
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*Use sample from [[Matt:LabNotes/2014-5-7]]
*Use sample from [[Matt:LabNotes/2014-5-7]]
**A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2-RevComp in one step
**A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2RevComp in one step
**D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2-RevComp after
**D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2RevComp after
**D5: Negative Ctrl (1uM dcProbe2-RevComp)
**D5: Negative Ctrl (1uM dcProbe2RevComp)


===Procedure===
===Procedure===
Line 17: Line 17:
| 2uM ppMALAT1||1.5
| 2uM ppMALAT1||1.5
|-
|-
| 100uM dcProbe2-RevComp||3
| 100uM dcProbe2RevComp||3
|-
|-
| H2O||265.5
| H2O||265.5
Line 51: Line 51:
| Total||100
| Total||100
|}
|}
*Aspirate D3&D4
*Add 100ul 1uM dcProbe2RevComp in 30% formamide + 2X SSC for 15min @ RT
**Preheated to 75C
*Aspirate ALL
*Wash with 2X SSC twice
*Image

Revision as of 01:44, 18 May 2014

Detecting ppMALAT1 Hybridization

  • Use sample from Matt:LabNotes/2014-5-7
    • A3, A4, & A5: Hybridize 10nM ppMALAT1 & 1uM dcProbe2RevComp in one step
    • D3 & D4: Hybridize 10nM ppMALAT1 for 24 hrs, 1uM dcProbe2RevComp after
    • D5: Negative Ctrl (1uM dcProbe2RevComp)

Procedure

  • Add 100ul of ppMALAT1 + dcProbe2-RevComp @45C for 24hrs (6:30p- )

A3-A5

Component Volume
10X Ampligase Buffer 30
2uM ppMALAT1 1.5
100uM dcProbe2RevComp 3
H2O 265.5
Total 300

D3&D4

Component Volume
10X Ampligase Buffer 20
2uM ppMALAT1 1
H2O 179
Total 200

D5

Component Volume
10X Ampligase Buffer 10
100uM dcProbe2RevComp 1
H2O 89
Total 100
  • Aspirate D3&D4
  • Add 100ul 1uM dcProbe2RevComp in 30% formamide + 2X SSC for 15min @ RT
    • Preheated to 75C
  • Aspirate ALL
  • Wash with 2X SSC twice
  • Image