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Team:Groningen/Notebook/Wetwork 4July2012
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(Created page with "{{HeaderGroningen2012}} '''Micro-array: RNA isolation and cDNA synthesis''' Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07)using: *Macaloïd method (see...") |
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'''Micro-array: RNA isolation and cDNA synthesis''' | '''Micro-array: RNA isolation and cDNA synthesis''' | ||
| - | 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: |
*Macaloïd method (see protocol [[File:Groningen_RR20120705_RNAisolation.pdf|here]]) | *Macaloïd method (see protocol [[File:Groningen_RR20120705_RNAisolation.pdf|here]]) | ||
| - | *Roche High pure RNA isolotion (see protocol [[File:Groninge_RR20120705_RNAisolation.pdf|here]] | + | *Roche High pure RNA isolotion (see protocol [[File:Groninge_RR20120705_RNAisolation.pdf|here]]) |
| - | Quality check with Agilent bio-analyser ([[File:Groningen__RR20120705_agilent|protocol.pdf]] and nanodrop ([[File:Groningen_RR20120705_nanodrop.pdf|protocol]] | + | Quality check with Agilent bio-analyser ([[File:Groningen__RR20120705_agilent|protocol.pdf]] and nanodrop ([[File:Groningen_RR20120705_nanodrop.pdf|protocol]]) |
Results nanodrop: | Results nanodrop: | ||
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*Fresh meat - 03/07: 4353,9 ng/ul; 260/280 = 1,54 | *Fresh meat - 03/07: 4353,9 ng/ul; 260/280 = 1,54 | ||
*Bad meat - 03/07: 3630,2 ng/ul; 260/280 = 1,96 | *Bad meat - 03/07: 3630,2 ng/ul; 260/280 = 1,96 | ||
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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: | ||
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*on ice | *on ice | ||
| - | + | Further with Reverse transcription using the Superscript III reverse transcriptase kit ([[File:Groningen_RR20120705_cDNAprotocol.pdf|protocol]]) | |
*18 hours, 42 degrees Celsius (O/N) | *18 hours, 42 degrees Celsius (O/N) | ||
Revision as of 12:20, 5 July 2012
Micro-array: RNA isolation and cDNA synthesis
Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07) using:
- Macaloïd method (see protocol File:Groningen RR20120705 RNAisolation.pdf)
- Roche High pure RNA isolotion (see protocol here)
Quality check with Agilent bio-analyser (protocol.pdf and nanodrop (protocol)
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 (File:Groningen RR20120705 cDNAprotocol.pdf)
- 18 hours, 42 degrees Celsius (O/N)
