Matt:LabNotes/2017-7-6

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Revision as of 22:16, 6 July 2017 by >Mzcai (→‎Samples)
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DARTFISH SplintR with 5% formamide or 10% DMF: 3 Probes

Samples

  • 10um sections cut by Yun on 4/11/2017
  • 3 samples
    1. No additives
    2. 5% formamide
    3. 10% dimethylformamide
  • 5% Gel mix with 1% crosslinker (normally 0.5%) and protocol is same as this with Acryloyl-X
Component Volume
40% AB 99:1 Mix 6.25
10% BSA 1
10mg/ml Acryloyl-X, SE in DMSO 1
H2O 39.75
5% TEMED 1
5% APS 1
Total 50

Protocol

Day 1

  1. Prepare 3 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Made 4% PFA in 1X PBS 1 week ago stored at RT
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
  3. Take out brain section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  7. Wash with cold nf-H2O three times and check for degradation
  8. Skip pepsin step usually done here
  9. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  10. Add 50ul gel casting mix filtered and degassed
  11. Seal in plastic bag and vacuum out air before filling with argon
  12. Let sit at RT for 30min
  13. Aspirate non-polymerized gel and wash once with 1X PBS
  14. Attach coverslip to bottom of petri dish
  15. Aspirate non-polymerized gel and wash once with 1X PBS

Prepare Padlock Probes

  • 1ul 10uM (50nM) each padlock probe
    • ppCUX2 (dcProbe6-Cy3)
      • /5Phos/TTCACCCTCTTGGTGATGGAGTAC CTTCAGCTTCCCGATATC TCGTAACCCGTGCGAAGTGC CTAGATTGTTGTCTGTGAGGACCTCC
    • ppBCL11B (dcProbe6-488)
      • /5Phos/AGCAGCTCAGTTTGCATTGCTT CTTCAGCTTCCCGATATC CACGCTTACGATCCCGCTAT TTGTTTCACAATTTCTCAAGGACTAA
    • ppMALAT1 (dcProbe2-Cy3)
      • /5Phos/TTTCTGCCTTTACTTATCAATTC CTTCAGCTTCCCGATATC CGACGGT CTACTTCGTCGCGTCAGACC AAATGGAGGTATGACATATAATCT
  • 20ul 10X (1X) SplintR Buffer
  • 10ul (5%) formamide or 20ul (10%) DMF or 0ul
  • 162ul H2O, 152ul H2O, or 172ul H2O
  • Pre-heat to 95C and then add to dish
  • Add 5ul 40U/ul (1U/ul) RNase Inhibitor
  • Put at 60C in EZ oven and set to 55C to incubate overnight