Hosuk:LabNotes/2013-11-7

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Revision as of 18:03, 15 November 2013 by >Hosuki78
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Padlock Probe Hybridization Test

Procedure

  • Padlock probes : ACTB, RAB7A, and MALAT1
    • Each concentration of Padlock probe were
    • 200uM in stock, use 2uM in 200uL mix when adding a single padlock probe
    • For 3 padlock probes, we mixed 67uL from each padlock probes to make 200uL, so each padlock probe concentration became 0.67uM
  • Detection probes
    • dcProbe1-Cy3 --> this does not work for padlock probe, but for 2nd Rolony detection, so we will not use this.
    • Shifted FISSEQ_2ndRCAprimerDye  has universal seq so it works for detection of all three padlocks
  • Each step
    • Step1 : ppRAB7A annealing : pre-heat @80C, add, incubate dish in oven @50C for 10min. Then add dcProbe1-Cy3, imaging
    • Step2 : Strip
    • Step3 : ppRAB7A+ppACTB+ppMALAT1 : pre-heat @80C, add, incubate dish in oven @50C for 30min. Then add Shifted FISSEQ_2ndRCAprimerDye, imaging
    • Step4 : Strip
    • Step5 : ppMALAT1 : pre-heat @80C, add, incubate dish in oven @50C for 10min. Then add Shifted FISSEQ_2ndRCAprimerDye imaging
    • Step6 : add Cy5-1st Rolony detection probe without stripping


Result

  • Further analysis will be done
  • Pre-heating @ 80C and incubate @50C for 10 min (for 1 padlock probe) or 30min (for 3 padlock probe) worked well.
    • will test lower concentration of pp.
  • MALAT1 gene were many!!
  • After adding Cy5-1st Rolony detection probe, MALAT1 signal became so much weaker, and number of MALAT1 lower.