Team:Heidelberg/Notebook measure/NotebookRT

From 2009.igem.org

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__NOTOC__
__NOTOC__
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== '''RT-PCR''' ==
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== '''Real-time RT-PCR''' ==
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== Contents ==
== Contents ==
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== 9-04-2009==
== 9-04-2009==
*RNA extration of HeLa transfected with CMV and JeT.
*RNA extration of HeLa transfected with CMV and JeT.
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**yield: 3.7 µg/1.5*10<sup>5</sup> cells
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[[Image:HD09_RNA_GEL1.png|thumb|left|900px|<div style="text-align:justify;">'''Gel electrophoresis of RNA extracted from HeLa cells.'''  1% agarose gel, 100V, 60 min. </div>]]
*Real-time RT PCR
*Real-time RT PCR
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**result:mRNA(CMV):mRNA(JeT)=1.8:1
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**result:mRNA(CMV):mRNA(JeT)=1.73:1
== 9-10-2009==
== 9-10-2009==
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*Dilution: to 20 ng/µl
*Dilution: to 20 ng/µl
*gel electrophoresis: positive
*gel electrophoresis: positive
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[[Image:HD09_RNA_GEL2.png|thumb|left|900px|<div style="text-align:justify;">'''Gel electrophoresis of RNA extracted from HeLa cells.'''  1% agarose gel, 100V, 60 min. </div>]]
== 9-25-2009==
== 9-25-2009==
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**Dilute the products to 20 ng/µl
**Dilute the products to 20 ng/µl
*Real-time RT PCR for CMV and JeT (50h)
*Real-time RT PCR for CMV and JeT (50h)
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Latest revision as of 19:24, 21 October 2009

Real-time RT-PCR

Contents

Week Days
35 8-24-2009 - 8-26-2009 8-27-2009 - - -
36 8-31-2009 - 9-02-2009 9-03-2009 9-04-2009 - -
37 - - - 9-10-2009 - - -
39 9-21-2009 - - 9-24-2009 9-25-2009 - -
40 - - 9-30-2009 - 10-02-2009 - -

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.
    • yield: 3.7 µg/1.5*105 cells
Gel electrophoresis of RNA extracted from HeLa cells. 1% agarose gel, 100V, 60 min.
  • Real-time RT PCR
    • result:mRNA(CMV):mRNA(JeT)=1.73:1

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
Gel electrophoresis of RNA extracted from HeLa cells. 1% agarose gel, 100V, 60 min.

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"