Matt:LabNotes/2014-3-4: Difference between revisions

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>Mzcai
(Created page with "==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 [[M...")
 
>Mzcai
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| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| 10X Ampligase||20
| 10X Ampligase Buffer||20
|-
|-
| 200uM ppMALAT1||2
| 200uM ppMALAT1||2
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| 200uM ppRAB7A||2
| 200uM ppRAB7A||2
|-
|-
| 5U/ul||20
| 5U/ul Ampligase||20
|-
|-
| H2O||154
| H2O||154

Revision as of 19:23, 4 March 2014

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
    • 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