Team:Heidelberg/Notebook measure/NotebookRT

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Contents

Notebook Real-time RT-PCR

Contents

8-24-2009

  • Design primers and probes for housekeeping genes (GAPDH, G6PD, 18s rRNA, TUBG2, ATCB) and fluorescent proteins (eGFP, mCherry).

8-26-2009

  • RNA extraction test (manually).
    • material and methods:
      • Sample: HeLa cells: 106 cells/tube
      • RNeasy Plant Mini Kit
      • Protocol: Purification of Total RNA from Animal Cells Using Spin Technology
    • Predicted yield (see "RNeasy Mini Handbook"-Qiagen): 15 µg/106 cells
    • Experimental yield (acquired with Nanodrop):
      • sample 1: 302 ng/µl, 18.12 µg/106 cells
      • sample 2: 181 ng/µl, 10.86 µg/106 cells

8-31-2009

  • RNA extraction (QIAcube)
    • material and methods:
      • Sample: HeLa cells (20 hours after transfection): 0.8*104 cells/tube
      • QIAcube
      • RNeasy Plant Mini Kit
      • Protocol: RNeasy Mini Animal Cell QIAshredder Version 2
    • Yield: not detectable for Nanodrop

9-02-2009

  • Primers and probes diluted to 100 µM
  • RNA extraction (QIAcube)
    • material and methods:
      • Sample: HeLa cells (with and without DMSO): 106 cells/tube
      • QIAcube
      • RNeasy Plant Mini Kit
      • Protocol: RNeasy Mini Animal Cell QIAshredder Version 2
    • Yield:
      • Sample 1 (without DMSO): 327.7 ng/µl, 16.39 µg/106 cells
      • Sample 2 (with DMSO): 56.2 ng/µl, 2.31 µg/106 cells

9-03-2009

  • Primers diluted to 5 µM
  • test PCR of primers

9-04-2009

  • RNA extration of HeLa transfected with CMV and JeT.
  • Real-time RT PCR
    • result:mRNA(CMV):mRNA(JeT)=1.8:1

9-10-2009

  • RNA extraction of Standard from HeLa cells using QIAcube
    • yield: 29 µg/2.4*106 cells
    • dilution: 20 ng/µl

9-21-2009

  • RNA extraction of HeLa cells using QIAcube
    • 12 samples, 1.5*105 cells/tube
    • Yield: <10 ng/µl
    • gel electrophoresis: negative

9-24-2009

  • RNA extraction of HeLa cells transfected with CMV and JeT using QIAcube
    • 12 samples each, 6.6*105 cells/tube
    • yield: CMV: 247.9 ng/µl, JeT: 238.3 ng/µl
  • Dilution: to 20 ng/µl
  • gel electrophoresis: positive

9-25-2009

  • real-time Reverse Transcription PCR for CMV and JeT (20h)

9-30-2009

  • RNA extraction of HeLa cells 50 hours after transfected with CMV and JeT

10-02-2009

  • NanoDrop the RNA extraction products
    • Dilute the products to 20 ng/µl
  • Real-time RT PCR for CMV and JeT (50h)
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