Hosuk:LabNotes/2014-8-10: Difference between revisions

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>Hosuki78
(Created page with "*LabNote ===Cancer Mutation detection=== ====Sample==== *Sample **NCIH1975_S1_2014-08-05 : -dUTP, rHex  45C **NCIH1975_S2_2014-08-03 : +dUTP, rHex  ...")
 
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===Cancer Mutation detection===
===Cancer Mutation detection===
====Sample====
====Decoding, and Analyzing====
*Sample
*Images
**NCIH1975_S1_2014-08-05 : -dUTP, rHex  45C
**ch00 - dcProbe[#]-488
**NCIH1975_S2_2014-08-03 : +dUTP, rHex  60C --> will use this later!
**ch01 - dcProbe[#]-Cy3
**ch02 - Bright field


*Steps
**Step1 : dcProbe0-488
**Step2 : dcProbe0-Cy3
**Step3 : dcProbe1-488
**Step4 : dcProbe1-Cy3
**...
**Step9 : dcProbe4-488
**Step10 : dcProbe4-Cy3


====Run Ampligase + Klentaq and 2nd RCA====
*Reaction Mix
[[File:Ampligase_Reagent_2014-08-07.jpg|600px]]


*I've taken images from 3 different positions (Pos1, Pos2, and Pos3)
**However the result that successfully analyzed was from only Pos1


*Start Ampligase+Klentaq : 08/05, 3:30pm (Just use NCIH1975_S1_2014-08-05), ~21.5hr at 45C
*Start RCA : 08/06, 2:40pm
**1uM ppRCA in 200uL 2x SSC, add and incubate at 42C for 36min,
**Wash with 2x SSC twice, 1x SSC twice, 1x PBS twice, then add Phi29 mix
*Finish : 08/07 (RCA for  ~18.5hr)




====Decoding====
*Procedure
*Decoding – 08/07
*#Figure out the offset pixels with BF image
*Sample : NCIH1975_S1_2014-08-05 : -dUTP, rHex
**Pos1
*1st Rolony – Cy3 : 1uM
**Standard : Step3
*dcProbe0~4 : 488, Cy3 : 1uM
**Step1 : X = 1, Y = -25
**dcProbe[#]-488 and dcProbe[#]-Cy3 are mixed in a sampe 100uL tube
**Step2 : X = 0, Y = -8
**Step3 : X = 0, Y = 0
**Step4 : X = -8, Y = 2
**Step5 : X = -8, Y = 9




*Step
*#Make maximum intensity projection(MIP) images at each step
*#Add dcProbe[#], incubate for 10min, image
*#Strip with 80% formamide(pre-heated at 70C) for 15min, wash with PBS twice
*#Repeat applying dcProbe and imaging
 
 
*Result : under anaylzinging

Revision as of 23:11, 12 August 2014

Cancer Mutation detection

Decoding, and Analyzing

  • Images
    • ch00 - dcProbe[#]-488
    • ch01 - dcProbe[#]-Cy3
    • ch02 - Bright field
  • Steps
    • Step1 : dcProbe0-488
    • Step2 : dcProbe0-Cy3
    • Step3 : dcProbe1-488
    • Step4 : dcProbe1-Cy3
    • ...
    • Step9 : dcProbe4-488
    • Step10 : dcProbe4-Cy3


  • I've taken images from 3 different positions (Pos1, Pos2, and Pos3)
    • However the result that successfully analyzed was from only Pos1


  • Procedure
    1. Figure out the offset pixels with BF image
    • Pos1
    • Standard : Step3
    • Step1 : X = 1, Y = -25
    • Step2 : X = 0, Y = -8
    • Step3 : X = 0, Y = 0
    • Step4 : X = -8, Y = 2
    • Step5 : X = -8, Y = 9


    1. Make maximum intensity projection(MIP) images at each step