Matt:LabNotes/2015-3-31

From ZhangLabWiki
Revision as of 01:36, 31 March 2015 by >Mzcai (Created page with "==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 ...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

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 2 4
2X Taq Master Mix 100 200
H2O 98 196
Total 200 400
  • 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
  • Add 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