Hosuk:LabNotes/2014-3-13: Difference between revisions

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====Experiment====
*8 Ampligase reaction,  
*8 Ampligase reaction,  
*4 NTC(no Ampligase emzyme)
*4 NTC(no Ampligase emzyme)
Line 36: Line 37:
**ppEGRF_L858R_MU 2uL
**ppEGRF_L858R_MU 2uL
**H2O 172 uL
**H2O 172 uL
====Result : 1st Rolonies====
*Number of rolonies from most of wells are not bad.
*PGP1F has more 1st rolonies than cancer cell has.
**I think main reason is the size of PGP1F is much more than cancer cell's, which make a big different.
**That means the density per '''area''' of rolonies seems similar at each cell type.
[[File:Mix_PGP1F_NCIH1975_Counts.png|300px]]
[[File:Mix_PGP1F_NCIH1975_Well04.png|300px]]
[[File:Mix_PGP1F_NCIH1975_Well06.png|300px]]
[[File:Mix_PGP1F_NCIH1975_Well10.png|300px]]

Revision as of 22:41, 14 March 2014

Mutation Detection with Mixed with PGP1F and NCIH1975 : Ampligase, 2nd RCA


Experiment

  • 8 Ampligase reaction,
  • 4 NTC(no Ampligase emzyme)

File:Mix PGP1F NCIH1975 24wellPlates AmpligaseMap.png


  • Procedure
    1. Make Ampligase mix, add
    2. Incubate at 45C for 24hr. (03/13 ~ 03/14)
    3. Run RCA reaction for 24hr. (03/14 ~ 03/15)
    4. Anneal dcProbes, and Image


  • Ampligase Mix
    • 10x Buffer 20uL
    • Ampligase (5U/uL) 8uL
    • ppEGRF_T790M_WT 2uL
    • ppEGRF_T790M_MU 2uL
    • ppEGRF_L858R_WT 2uL
    • ppEGRF_L858R_MU 2uL
    • H2O 164 uL


  • NTC Mix
    • 10x Buffer 20uL
    • Ampligase (5U/uL) 0uL
    • ppEGRF_T790M_WT 2uL
    • ppEGRF_T790M_MU 2uL
    • ppEGRF_L858R_WT 2uL
    • ppEGRF_L858R_MU 2uL
    • H2O 172 uL


Result : 1st Rolonies

  • Number of rolonies from most of wells are not bad.
  • PGP1F has more 1st rolonies than cancer cell has.
    • I think main reason is the size of PGP1F is much more than cancer cell's, which make a big different.
    • That means the density per area of rolonies seems similar at each cell type.

File:Mix PGP1F NCIH1975 Counts.png


File:Mix PGP1F NCIH1975 Well04.png


File:Mix PGP1F NCIH1975 Well06.png


File:Mix PGP1F NCIH1975 Well10.png