AlanFung:LabNotes/Rolony/2014-3-5: Difference between revisions

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===Anneal Padlock Probes for 24 hours===
===Anneal Padlock Probes for 24 hours===
#Wash twice w PBS
#1X Ampligase Buffer (10ul of stock 10X)
#1X Ampligase Buffer (10ul of stock 10X)
#Add padlock probe to each well with their corresponding concentration (35ul)
#Add padlock probe to each well with their corresponding concentration (35ul)

Revision as of 21:16, 7 March 2014

Secondary Rolony Generation for Padlock Probe Concentration Test

Samples

  • 96 well plate containing NCIH-1975 cells fixed by Hosuk
  • Location and save location of 12 position in 96 well plate for the experiment
  • High Concentration (100pM)
  • Low Concentration (1pM)
  • Ultra Low Concentration (0.1pM)

Fixing Template in cells

  1. Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (60ul 100uM MALAT1_Template + 60ul 20X SSC + 480ul H2O)
  2. Add 50ul to each well
    • Incubate @RT for 15min
  3. Aspirate but don't wash
  4. Add 50ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS)
    • Incubate @RT for 1hr
  5. Wash with 1X PBS twice
  6. Add 50ul 1M Tris pH 8.0
    • Incubate @ RT for 30min

Dilute 2nM ppMALAT1

  • 100pM
  • 1pM
  • 0.1pM

Anneal Padlock Probes for 24 hours

  1. Wash twice w PBS
  2. 1X Ampligase Buffer (10ul of stock 10X)
  3. Add padlock probe to each well with their corresponding concentration (35ul)
  4. 0.5 U/ul Ampligase (5ul of stock 5U/ul)
  5. Incubate @ 45C for 24 hours
  6. Wrapped in parafilm and put in ziploc with wet towel

Group 1 100pM ppMALAT1

Group 1   Group 2   Group 3
Image Well Mark & Find   Image Well Mark & Find   Image Well Mark & Find
A C3 1   E F8 15   I C6 9
B D3 2   F F5 14   J C7 10
C F3 13   G F6 7   K E7 11
D C5 4   H E6 8   L F7 12
  1 2 3 4 5 6 7 8 9 10 11 12
A                        
B                        
C     C3   C5 C6 C7          
D     D3                  
E           E6 E7          
F     F3   F5 F6 F7 F8        
G                        
H                        

File:Ppconcentration NCHI1975 Group1.jpg

File:Ppconcentration NCHI1975 Group2.jpg

File:Ppconcentration NCHI1975 Group3.jpg