Matt:LabNotes/2014-2-25: Difference between revisions

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>Mzcai
>Mzcai
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====Detection====
====Detection====
*To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C)
*To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C)
**Strip with 80% formamide
**Strip E6 & F6 with 80% formamide @ 37C
*To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
*To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
**Strip with 80% formamide
**Strip with 80% formamide @37C
*To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
*To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
**Strip with 80% formamide

Revision as of 03:32, 28 February 2014

Secondary Rolony Generation

Samples: Matt:LabNotes/2014-2-21

Procedure

Anneal Padlock Probes

  • 1X Ampligase Buffer
  • 2uM of each padlock probe
  • 0.5 U/ul Ampligase
  • Incubate @ 45C for 24 hours
    • Wrapped in parafilm and put in ziploc with wet towel
Fixed Template - 25U Ampligase (C9 & F9)
Component Volume
10X Ampligase Buffer 10
200uM ppMALAT1 1
5U/ul Ampligase 10
H2O 79
Total 100
Fixed Template - 50U Ampligase (C10 & F10)
Component Volume
10X Ampligase Buffer 10
200uM ppMALAT1 1
5U/ul Ampligase 20
H2O 69
Total 100
1st Rolony (E6 & F6)
Component Volume
10X Ampligase Buffer 10
200uM ppMALAT1 1
200uM ppACTB 1
200uM ppRAB7A 1
5U/ul Ampligase 10
H2O 77
Total 100
Empty Negative Control (D7 & E7)
  • Not going to waste Ampligase since it's highly unlikely anything will be ligated, but will do RCA in these wells
Pre-circularized Padlock Probes (C7 & C8)
  • Combined all the pre-circle ppMALAT1 made on: Matt:LabNotes/2014-2-10 into 64ul
  • 75% EtOH for 10 minutes
  • Wash with 1X PBS 2 times
  • 0.1N HCl for 2 minutes
  • Wash with 1X PBS 3 times
  • Add 32ul to C7 & C8
Fixed Pre-circularized Padlock Probes (F7 & F8)
  • Not going to do anything during Ampligase step

Secondary RCA

  1. Aspirate each well but don't wash
    • F7 well was dried up
  2. Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
    1. 3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 50ul to each well, incubate at 45C for 15min.
    4. Aspirate, and wash using 2x SSC twice
    5. Aspirate, and wash using 0.1x SSC twice
  3. Prepare the rolling circle amplification reaction mixtures on ice.
    1. 513 ul ddH2O,
    2. 60 ul 10x Phi29 buffer,
    3. 6 ul 25 mM dNTP,
    4. 12 ul 2 mM aminoallyl dUTP
    5. 9 ul Phi29 DNA polymerase (Epicentre, low concentration)
      • 600ul Total Volume
  4. Add 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14)
  5. Wash 1X PBS once
  6. Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS)
  7. Wash 1X PBS twice
  8. Add 50ul 1M Tris pH 8.0 @RT for 1hr
  9. Wash 1X PBS twice

Detection

  • To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C)
    • Strip E6 & F6 with 80% formamide @ 37C
  • To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
    • Strip with 80% formamide @37C
  • To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)