Team:Heidelberg/Notebook measure/NotebookRT

From 2009.igem.org

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='''Notebook Real-time RT-PCR'''=
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== RT-PCR ==
== Contents ==
== Contents ==

Revision as of 11:43, 20 October 2009

RT-PCR

Contents

Week Days
33 - 8-11-2009 - - - - -
34 - 8-18-2009 8-19-2009 8-20-2009 8-21-2009 - -
35 8-24-2009 8-25-2009 8-26-2009 8-27-2009 8-28-2009 8-29-2009 -
36 8-31-2009 9-01-2009 9-02-2009 9-03-2009 9-04-2009 - -
37 9-07-2009 9-08-2009 9-09-2009 9-10-2009 9-11-2009 - -
38 9-14-2009 9-15-2009 9-16-2009 9-17-2009 9-18-2009 - 9-20-2009
39 9-21-2009 9-22-2009 9-23-2009 9-24-2009 9-25-2009 9-26-2009 -
40 9-28-2009 9-29-2009 - - - - -

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)
Retrieved from "http://2009.igem.org/Team:Heidelberg/Notebook_measure/NotebookRT"