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Team:Heidelberg/Notebook measure/NotebookRT
From 2009.igem.org
Revision as of 09:24, 20 October 2009 by
Chenchenzhu
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Contents
1
Notebook Microscopy
1.1
Contents
1.2
8-24-2009
1.3
8-26-2009
1.4
8-31-2009
1.5
9-02-2009
1.6
9-03-2009
1.7
9-04-2009
1.8
9-10-2009
1.9
9-21-2009
1.10
9-24-2009
1.11
9-25-2009
1.12
9-30-2009
1.13
10-02-2009
Notebook Microscopy
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: 10
6
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/10
6
cells
Experimental yield (acquired with Nanodrop):
sample 1: 302 ng/µl, 18.12 µg/10
6
cells
sample 2: 181 ng/µl, 10.86 µg/10
6
cells
8-31-2009
RNA extraction (QIAcube)
material and methods:
Sample: HeLa cells (20 hours after transfection): 0.8*10
4
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): 10
6
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/10
6
cells
Sample 2 (with DMSO): 56.2 ng/µl, 2.31 µg/10
6
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*10
6
cells
dilution: 20 ng/µl
9-21-2009
RNA extraction of HeLa cells using QIAcube
12 samples, 1.5*10
5
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*10
5
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)