Hosuk:LabNotes/2013-9-12: Difference between revisions

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*Prepare two barcoded padlock probes - ACTB, RAB7A
*Prepare two barcoded padlock probes - ACTB, RAB7A


=====for detecting ACTB=====
=====Padlock probe for ACTB=====
*ppACTB : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp
*'''ppACTB''' : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp
**two arms : CTGTGCTCGCGGGGCG, GGCAAAGGCGAGGCT
**two arms : CTGTGCTCGCGGGGCG, GGCAAAGGCGAGGCT
**site for decoding probes (dcProbe0-Cy3) : ACGTATCGGTAGTCGCAACGCA --> 22bp
**site for decoding probes (dcProbe0-Cy3) : ACGTATCGGTAGTCGCAACGCA --> 22bp
**site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp
**site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp


=====for detecting RAB7A=====
=====Padlock probe for RAB7A=====
*ppRAB7A: /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp
*'''ppRAB7A''' : /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp
**two arms : GAAGCGAGAAGGTCCAAGTTCTG, GAGGAGACTAAACGGAGGACA  
**two arms : GAAGCGAGAAGGTCCAAGTTCTG, GAGGAGACTAAACGGAGGACA  
**site for decoding probes (dcProbe1-Cy3) : GTCTTGCGTGCGATACGGAGTA --> 22bp
**site for decoding probes (dcProbe1-Cy3) : GTCTTGCGTGCGATACGGAGTA --> 22bp
**site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp
**site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp


=====FISSEQ_ppRCA=====
=====2nd RCA probe=====
*GATATCGGGAAGCTGA*A*G --> 18bp
*'''FISSEQ_ppRCA''' : GATATCGGGAAGCTGA*A*G --> 18bp




*Start at 09/10
*Start at 09/10

Revision as of 17:58, 12 September 2013

Gene decoding with Barcoded Padlock probes

Experimental plan

  • Hybridizing padlock probes on 1st rolonies, and
  • Generate 2nd rolonies over 1st rolonies, and
  • Observe each different fluorescent signal from 2nd rolonies


Detail procedure

  • Prepare two barcoded padlock probes - ACTB, RAB7A
Padlock probe for ACTB
  • ppACTB : /5Phos/CTGTGCTCGCGGGGCG CTTCAGCTTCCCGATATC CGACGG ACGTATCGGTAGTCGCAACGCA GGCAAAGGCGAGGCT --> 77bp
    • two arms : CTGTGCTCGCGGGGCG, GGCAAAGGCGAGGCT
    • site for decoding probes (dcProbe0-Cy3) : ACGTATCGGTAGTCGCAACGCA --> 22bp
    • site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp
Padlock probe for RAB7A
  • ppRAB7A : /5Phos/GAAGCGAGAAGGTCCAAGTTCTG CTTCAGCTTCCCGATATC CGACGG GTCTTGCGTGCGATACGGAGTA GAGGAGACTAAACGGAGGACA --> 90bp
    • two arms : GAAGCGAGAAGGTCCAAGTTCTG, GAGGAGACTAAACGGAGGACA
    • site for decoding probes (dcProbe1-Cy3) : GTCTTGCGTGCGATACGGAGTA --> 22bp
    • site for 2nd RCA probes : CTTCAGCTTCCCGATATC --> 18bp
2nd RCA probe
  • FISSEQ_ppRCA : GATATCGGGAAGCTGA*A*G --> 18bp


  • Start at 09/10