Team:Groningen/Notebook/Wetwork 4July2012

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'''Micro-array: RNA isolation and cDNA synthesis'''
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Micro-array: RNA isolation and cDNA synthesis<br><br>
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Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07)using:
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Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07) using:<br>
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*Macaloïd method (see protocol [[File:Groningen_RR20120705_RNAisolation.pdf|here]])
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*Macaloïd method <br>
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*Roche High pure RNA isolotion (see protocol [[File:Groninge_RR20120705_RNAisolation.pdf|here]]
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*Roche High pure RNA isolotion <br><br>
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Quality check with Agilent bio-analyser ([[File:Groningen__RR20120705_agilent|protocol.pdf]] and nanodrop ([[File:Groningen_RR20120705_nanodrop.pdf|protocol]]
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Quality check with Agilent bio-analyser and nanodrop <br>
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Results nanodrop:
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Results nanodrop:<br>
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*Fresh meat - 02/07: 4458,6 ng/ul; 260/280 = 1,09
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*Fresh meat - 02/07: 4458,6 ng/ul; 260/280 = 1,09<br>
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*Bad meat - 02/07: 3667,0 ng/ul; 260/280 = 1,97
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*Bad meat - 02/07: 3667,0 ng/ul; 260/280 = 1,97<br>
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*Fresh meat - 03/07: 4353,9 ng/ul; 260/280 = 1,54
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*Fresh meat - 03/07: 4353,9 ng/ul; 260/280 = 1,54<br>
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*Bad meat - 03/07: 3630,2 ng/ul; 260/280 = 1,96
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*Bad meat - 03/07: 3630,2 ng/ul; 260/280 = 1,96<br><br>
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cDNA synthesis (in duplo (named a/b in the rest of the notebook)) using the following mixture:
 
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*Fresh meat - 02/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers
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cDNA synthesis (in duplo (named a/b in the rest of the notebook)) using the following mixture:<br>
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*Bad meat - 02/07: 4,1 ul sample; 11,9 ul H20; 2 ul random nonamers
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*Fresh meat - 03/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers
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*Bad meat - 03/07: 4,2 ul sample; 11,8 ul H20; 2 ul random nonamers
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*mixed, 5' 70 degrees Celsius.
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*Fresh meat - 02/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers<br>
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*10 min RT
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*Bad meat - 02/07: 4,1 ul sample; 11,9 ul H20; 2 ul random nonamers<br>
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*on ice
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*Fresh meat - 03/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers<br>
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*Bad meat - 03/07: 4,2 ul sample; 11,8 ul H20; 2 ul random nonamers<br> <br>
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*Further with Reverse transcription using the Superscript III reverse transcriptase kit ([[File:Groningen_RR20120705_cDNAprotocol.pdf|protocol]])
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*mixed, 5' 70 degrees Celsius.<br>
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*18 hours, 42 degrees Celsius (O/N)
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*10 min RT<br>
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*on ice<br><br>
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Further with Reverse transcription using the Superscript III reverse transcriptase kit <br>
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*18 hours, 42 degrees Celsius (O/N)<br><br>
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Emeraldo/Nisa<br><br>
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1.) B. subtilis transformation failed (as reported by Ruud)<br>
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2.) Our BBa_K090403 is not good. Find alternatice source of this biobrick!<br>
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3.) Ask about B. subtilis transformation from Edinburgh 2007, the team that submitted BBa_I742123<br>
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4.) Digestion of BBa_I742123, BBa_K274100 (red pigment), and BBa_K274100 (voilet pigment) with SpeI and EcoRI for later ligation to backbone.<br>
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<br>
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<A HREF="https://2012.igem.org/Team:Groningen/Notebook"><FONT COLOR=#ff6700>Back to notebook</FONT></A>
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{{Template:SponsorsGroningen2012}}

Latest revision as of 16:56, 25 September 2012








Micro-array: RNA isolation and cDNA synthesis

Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07) using:
*Macaloïd method
*Roche High pure RNA isolotion

Quality check with Agilent bio-analyser and nanodrop
Results nanodrop:
*Fresh meat - 02/07: 4458,6 ng/ul; 260/280 = 1,09
*Bad meat - 02/07: 3667,0 ng/ul; 260/280 = 1,97
*Fresh meat - 03/07: 4353,9 ng/ul; 260/280 = 1,54
*Bad meat - 03/07: 3630,2 ng/ul; 260/280 = 1,96

cDNA synthesis (in duplo (named a/b in the rest of the notebook)) using the following mixture:
*Fresh meat - 02/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers
*Bad meat - 02/07: 4,1 ul sample; 11,9 ul H20; 2 ul random nonamers
*Fresh meat - 03/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers
*Bad meat - 03/07: 4,2 ul sample; 11,8 ul H20; 2 ul random nonamers

*mixed, 5' 70 degrees Celsius.
*10 min RT
*on ice

Further with Reverse transcription using the Superscript III reverse transcriptase kit
*18 hours, 42 degrees Celsius (O/N)

Emeraldo/Nisa

1.) B. subtilis transformation failed (as reported by Ruud)
2.) Our BBa_K090403 is not good. Find alternatice source of this biobrick!
3.) Ask about B. subtilis transformation from Edinburgh 2007, the team that submitted BBa_I742123
4.) Digestion of BBa_I742123, BBa_K274100 (red pigment), and BBa_K274100 (voilet pigment) with SpeI and EcoRI for later ligation to backbone.

Back to notebook



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