Hosuk:LabNotes/2013-8-5

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Revision as of 00:22, 6 August 2013 by >Hosuki78
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Test Decoding probes using custom pre-circularized oligos

Previous try

  • Alan and I have tried decoding pre-circularized oligos made from Matt at 07/30/2013.
  • I added oligos in fixed cells on MatTek glass-bottom dish, and ran RCA to generate rolonies.
  • However the signal wasn’t seen any FL signal after hybridizing decoding probes.
  • We suspected that the amount of oligos might not enough (The actual concentration or amount of the oligos was unknown).
  • So I've ordered 90bp ssDNA from IDT which has 3 sites for decoding probes and 1 site for RCA probes.


2nd try with 90bp custom oligo

=Template design

  • Sequence : /5Phos/CCCGATATCCGACGGTAGTGTGTCTTGCGTGCGATACGGAGTATCTACTTCGTCGCGTCAGACCATCGGAATACGTCGTTGACTGCGTTC
  • RCA probe sequence : ACACTACCGTCGGATATC
  • Decoding probes for this experiment
    • dcProbe1-Cy3 : Cy3-GTCTTGCGTGCGATACGGAGTA
    • dcProbe2-Cy3 : Cy3-TCTACTTCGTCGCGTCAGACCA
    • dcProbe4-FAM : FAM-TCGGAATACGTCGTTGACTGCG

File:Sequence Template Probes.png


=Circularization

  • CircLigase II reaction with 50pmole input DNA
    • 6 hr reaction

File:CircLigaseII 20uL.png


  • Circ. reaction lane has no band near 90bp, it seemed circularized.

File:CircLigaseII 20uL Gel.png


  • Start at 06/29
    • Use cell dishes fixed at 07/07 --> Cells were not plenty… need to thaw new one
    • GC, lysine coated dish
    • Run RT : 5:00pm 07/07 ~ 9:00am 07/08 --> ~15hr.
    • Run CircLigase II : S1 --> 1:30pm 07/08 ~ 3:30pm 07/08 --> 2hr.
    • Run CircLigase II : S2 --> 11:30am 07/08 ~ 3:30pm 07/08 --> 4hr.
    • Run RCA : 4:30 pm 07/08 ~ 12:30pm 07/09 --> ~20hr.


Result