Matt:LabNotes/2014-1-15

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Exo I/III in situ to check 2ndary Rolony Ampligase reaction[edit]

  • Currently 2nd Rolony fabrication is inefficient or unsuccessful
    • Need a positive and negative control (currently working on making positive control by pre-circularizing ppMALAT1)
    • Trying to determine whether Ampligase reaction or 2nd RCA is the limiting step
  • How to separate Ampligase step from 2nd RCA step?
    • Add Exo I/III to cells after Ampligase step to digest non-circular DNA
    • Then can use Shifted FISSEQ_2ndRCAprimerDye to detect the padlock probes that are left (and therefore circularized)
    • By using positive and negative control, can determine yield of Ampligase step

Procedure[edit]

  • Use 6 wells with 1st Rolonies from 96-well plate:
  • 2 Positive control (C7 & D7): +Ampligase, -Exo (expect to see lots of signal like previous experiments)
  • 2 Negative control (E7 & F7): -Ampligase, +Exo (expect to see no signal)
  • 2 Experimental (D9 & E9): +Ampligase, +Exo (expect to see a % of a positive control, hopefully close to 100%)

Determine Number of ppMALAT1 targets

  1. Hybridize 2nM ppMALAT1 (in 2X SSC)
    1. Preheat to 70C for 5min and then add 50ul to each well
    2. Incubate 50C for 10min
    3. Wash once with 2X SSC
  2. Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC (206.5ul H2O + 105ul 100% formamide + 3.5ul 200uM Dye + 35ul 20X SSC = 350ul)
    1. Preheat to 60C for 5min and then add 50ul to each well
    2. Incubate RT for 15min
    3. Wash 2 times with 2X SSC
  3. Image (ATTO488)
  4. Strip with 80% formamide for 10min @50C

Ampligase Experiment

  1. Hybridize 2nM ppMALAT1 (in 2X SSC)
    1. Preheat to 70C for 5min and then add 50ul to each well
    2. Incubate 50C for 10min
    3. Wash once with 2X SSC
  2. Add 0.05U/ul Ampligase to Positive and Exprimental (2ul Ampligase + 20ul 10X Buffer + 178ul H2O)
    1. Negative control: (90ul H2O + 10ul 10X buffer)
    2. Incubate 50C for 4hrs
  3. Wash twice with 1X PBS
  4. Add 50ul Exo I/III to each well (2.5ul 20U/ul Exo I and 2.5ul 200U/ul Exo III)
    1. Exo I/III mix: 20ul 10X Exo III Buffer + 10ul Exo I + 10ul Exo III + 160ul H2O
    2. Positive control: 50ul of (10ul 10X Exo III Buffer + 90ul H2O)
    3. Incubate 37C for 2hrs
  5. Wash twice with 1X PBS preheated to 94C
    1. Let sit for 1min before aspirating
  6. Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC (206.5ul H2O + 105ul 100% formamide + 3.5ul 200uM Dye + 35ul 20X SSC = 350ul)
    1. Preheat to 60C for 5min and then add 50ul to each well
    2. Incubate RT for 15min
    3. Wash 2 times with 2X SSC
  7. Image (ATTO488)