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Team:Groningen/Notebook/Wetwork 4July2012
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| + | Micro-array: RNA isolation and cDNA synthesis<br><br> | ||
| - | Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07) using: | + | Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07) using:<br> |
| - | *Macaloïd method | + | *Macaloïd method <br> |
| - | *Roche High pure RNA isolotion | + | *Roche High pure RNA isolotion <br><br> |
| - | Quality check with Agilent bio-analyser | + | Quality check with Agilent bio-analyser and nanodrop <br> |
| - | Results nanodrop: | + | Results nanodrop:<br> |
| - | *Fresh meat - 02/07: 4458,6 ng/ul; 260/280 = 1,09 | + | *Fresh meat - 02/07: 4458,6 ng/ul; 260/280 = 1,09<br> |
| - | *Bad meat - 02/07: 3667,0 ng/ul; 260/280 = 1,97 | + | *Bad meat - 02/07: 3667,0 ng/ul; 260/280 = 1,97<br> |
| - | *Fresh meat - 03/07: 4353,9 ng/ul; 260/280 = 1,54 | + | *Fresh meat - 03/07: 4353,9 ng/ul; 260/280 = 1,54<br> |
| - | *Bad meat - 03/07: 3630,2 ng/ul; 260/280 = 1,96 | + | *Bad meat - 03/07: 3630,2 ng/ul; 260/280 = 1,96<br><br> |
| - | cDNA synthesis (in duplo (named a/b in the rest of the notebook)) using the following mixture: | + | cDNA synthesis (in duplo (named a/b in the rest of the notebook)) using the following mixture:<br> |
| - | *Fresh meat - 02/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers | + | *Fresh meat - 02/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers<br> |
| - | *Bad meat - 02/07: 4,1 ul sample; 11,9 ul H20; 2 ul random nonamers | + | *Bad meat - 02/07: 4,1 ul sample; 11,9 ul H20; 2 ul random nonamers<br> |
| - | *Fresh meat - 03/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers | + | *Fresh meat - 03/07: 3,4 ul sample; 12,6 ul H20; 2 ul random nonamers<br> |
| - | *Bad meat - 03/07: 4,2 ul sample; 11,8 ul H20; 2 ul random nonamers | + | *Bad meat - 03/07: 4,2 ul sample; 11,8 ul H20; 2 ul random nonamers<br> <br> |
| - | *mixed, 5' 70 degrees Celsius. | + | *mixed, 5' 70 degrees Celsius.<br> |
| - | *10 min RT | + | *10 min RT<br> |
| - | *on ice | + | *on ice<br><br> |
| - | Further with Reverse transcription using the Superscript III reverse transcriptase kit | + | Further with Reverse transcription using the Superscript III reverse transcriptase kit <br> |
| - | *18 hours, 42 degrees Celsius (O/N) | + | *18 hours, 42 degrees Celsius (O/N)<br><br> |
| - | Emeraldo/Nisa | + | Emeraldo/Nisa<br><br> |
| - | 1.) B. subtilis transformation failed (as reported by Ruud) | + | 1.) B. subtilis transformation failed (as reported by Ruud)<br> |
| - | 2.) Our BBa_K090403 is not good. Find alternatice source of this biobrick! | + | 2.) Our BBa_K090403 is not good. Find alternatice source of this biobrick!<br> |
| - | 3.) Ask about B. subtilis transformation from Edinburgh 2007, the team that submitted BBa_I742123 | + | 3.) Ask about B. subtilis transformation from Edinburgh 2007, the team that submitted BBa_I742123<br> |
| - | 4.) Digestion of BBa_I742123, BBa_K274100 (red pigment), and BBa_K274100 (voilet pigment) with SpeI and EcoRI for later ligation to | + | 4.) Digestion of BBa_I742123, BBa_K274100 (red pigment), and BBa_K274100 (voilet pigment) with SpeI and EcoRI for later ligation to backbone.<br> |
| + | <br> | ||
| + | <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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