Team:Groningen/Notebook/Wetwork 5July2012


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.

Qualitycheck with nanodrop.

*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


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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