Matt:LabNotes/2015-3-31: Difference between revisions

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>Mzcai
>Mzcai
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| align="center" style="background:#f0f0f0;"|'''2X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| 100uM SeqRolony_CL_Amplify||2||4
| 100uM SeqRolony_CL_Amplify||1||2
|-
|-
| 2X Taq Master Mix||100||200
| 2X Taq Master Mix||50||100
|-
|-
| H2O||98||196
| H2O||49||98
|-
|-
| Total||200||400
| Total||100||200
|}
|}
*Place cover-slip on clean glass slide (tissue face up)
*Place cover-slip on clean glass slide (tissue face up)
*Use rubber seal with circular opening to surround tissue
*Use rubber seal with circular opening to surround tissue
**Rubber has double-sided tape and use to create seal around the tissue
**Rubber has double-sided tape and use to create seal around the tissue
*Add PCR mix and seal with plastic to prevent evaporation (add mineral oil on top?)
***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
*Put on thermocycler with slide adapter
   '''Program''' 90C 3min -> (90C 20sec -> 58C 30sec -> 68C 45sec) x 20 -> 68C 2min -> 15C hold
   '''Program''' 90C 3min -> (90C 20sec -> 58C 30sec -> 68C 45sec) x 20 -> 68C 2min -> 15C hold

Revision as of 20:44, 31 March 2015

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