Matt:LabNotes/2014-3-4

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Revision as of 20:11, 4 March 2014 by >Mzcai (→‎Procedure)
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3 Genes Secondary Rolonies Mattek Dish

  • Hosuk got much more primary rolonies (incl. ACTB and RAB7A) by culturing PGP1F without EGF (Hosuk:LabNotes/2014-3-3)
  • Since Matt:LabNotes/2014-2-25#Detection resulted in many MALAT1 secondary rolonies but few ACTB or RAB7A
    • This is possibly due to sample having very few ACTB or RAB7A
    • Also possible the dyes we got from Illumina (dcProbe0-Cy3 and dcProbe1-Cy3) have somehow degraded since MALAT1 detection was using a new dye-probe from IDT

Procedure

  1. Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
  2. Wash with 1X PBS twice
  3. Anneal Padlock Probes @45C for 24hrs (Put in at noon, some bubbles and hard to keep on middle glass)
    • 1X Ampligase Buffer
    • 2uM of each padlock probe
    • 0.5 U/ul Ampligase
Component Volume
10X Ampligase Buffer 20
200uM ppMALAT1 2
200uM ppACTB 2
200uM ppRAB7A 2
5U/ul Ampligase 20
H2O 154
Total 200