Matt:LabNotes/2015-3-31

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Revision as of 20:51, 31 March 2015 by >Mzcai (→‎Add Sequencing Adapters)
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Sequence in situ Rolonies via in situ Slide PCR

  • Purpose: Sequence the rolonies in situ to quantify the contributions of each probe to the decoded rolonies
    • ie gene GNG4 has many rolonies but are they due to a single highly efficient probe or a number of different probes

Protocol

  • Samples:
    • Exp: BA8 CA12k_Nov2014_V4 Captured Rolonies
    • NegCtrl: BA8 1st (cDNA) Rolonies (from old FISSEQ protocol)

Strip hybridized dye-probes with 80% formamide pre-heated to 80C

  • The tissue is on thin cover-slip placed on a glass slide
  • Take cover-slip off glass slide and put in small culture dish
  • Incubate for 15min at RT and then wash twice with 1X PBS

Slide PCR

  • Make PCR Reaction Mix on ice
Components Volume 2X Volume
100uM SeqRolony_CL_Amplify 1 2
2X Taq Master Mix 50 100
H2O 49 98
Total 100 200
  • Place cover-slip on clean glass slide (tissue face up)
  • Use rubber seal with circular opening to surround tissue
    • Rubber has double-sided tape and use to create seal around the tissue
      • Had to use gap seal 200 glue to plug leaks
  • Add 100ul PCR mix and seal with plastic to prevent evaporation
  • Put on thermocycler with slide adapter
 Program 90C 3min -> (90C 20sec -> 58C 30sec -> 68C 45sec) x 20 -> 68C 2min -> 15C hold
  • Remove plastic seal and pipette solution into PCR tubes
  • Add 100ul H2O, let sit for 5min, and pipette into empty PCR tubes (to get more amplicon)

Add Sequencing Adapters

PCR Test

Components 1X Volume 3X Volume
Slide PCR amplicon 5 0
SeqRolony_P5 0.4 1.2
SeqRolony_P7 0.4 1.2
2X KAPA SYBG MM 12.5 37.5
H2O 6.7 20.1
Total 25 60
  • Aliquot 20ul from 3X master mix and add 5ul slide amplicon
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

[[ | 650px]]

PCR

Components 1X Volume 3X Volume
Slide PCR amplicon 5 0
SeqRolony_P5 0.4 1.2
SeqRolony_P7 0.4 1.2
2X KAPA SYBG MM 12.5 37.5
H2O 6.7 20.1
Total 25 60