Team:Heidelberg/Notebook measure/NotebookRT


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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
 * 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

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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