Team:Groningen/Notebook/Wetwork 4July2012

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'''Micro-array: RNA isolation and cDNA synthesis'''
'''Micro-array: RNA isolation and cDNA synthesis'''

Revision as of 19:01, 10 July 2012





Micro-array: RNA isolation and cDNA synthesis

Isolated RNA from the harvested cells (Badmeat/Freshmeat 02/07 & 03/07) using:

Quality check with Agilent bio-analyser (File:Groningen RR20120705 agilent.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)


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 backboe.

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