Team:Groningen/Notebook/Wetwork 5July2012

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'''Micro-array preparation'''
'''Micro-array preparation'''

Revision as of 19:01, 10 July 2012





Micro-array preparation

cDNA purification

degradation of mRNA:

  • added 3 ul of 2,5 M NaOH to reverse transcription mix (ran overnight)
  • vortexed, spinned down
  • 15'37 degrees Celsius
  • added 15 ul 2 M HEPES free acid
  • mixed by pipetting up/down

purified cDNA using NucleoSpin Extract II columns (File:Groningen RR20120705 cDNAprotocol.pdf).

Qualitycheck with nanodrop.

Results:

  • Fresh_0207A: 274,3 ng/ul
  • Fresh_0207B: 247,2 ng/ul
  • Bad_0207A: 232,9 ng/ul
  • Bad_0207B: 42,9 ng/ul ------> not enough (treshold = 60 ng/ul).
  • Fresh_0307A: 249,8 ng/ul
  • Fresh_0307B: 241,0 ng/ul (but low quality)
  • Bad_0307A: 249,8 ng/ul
  • Bad_0307B: 69,9 ng/ul

kept all cDNA in -80 freezer. repeated cDNA synthesis for Fresh_0307A, Bad_0207B, Bad_0307B


NEW POSSIBLE BACKBONE! Also for biobrick submission

Nisa/Emeraldo

1.) After discussion with Jan Willem about our critical situation in the backbone problem, we found new backbone that can be used for B. subtilis: integration plasmid with double crossover on SacA gene, cm resistant (B. subtilis) and amp+cm resistant in E. coli. We have to engineer this backbone to be biobrick ready by adding prefix and suffix + terminator in the MCS.

2.) O/N the E.coli containing the new plasmid(pSAC-CM) for plasmid isolation tomorrow.

3.) Preparation for B.substilis transformation: Molgen method and Team Edinburgh method; Tomorrow: transform B. subtilis with pSac-CM and BBa_I742123 with two different methods.

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