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Team:Groningen/Notebook/Wetwork 5July2012
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| - | + | Micro-array preparation<br><br> | |
| - | + | cDNA purification<br><br> | |
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| - | + | degradation of mRNA:<br> | |
| + | *added 3 ul of 2,5 M NaOH to reverse transcription mix (ran overnight)<br> | ||
| + | *vortexed, spinned down<br> | ||
| + | *15'37 degrees Celsius<br> | ||
| + | *added 15 ul 2 M HEPES free acid<br> | ||
| + | *mixed by pipetting up/down<br><br> | ||
| - | + | purified cDNA using NucleoSpin Extract II columns.<br><br> | |
| - | + | Qualitycheck with nanodrop.<br><br> | |
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| - | kept all cDNA in -80 freezer. repeated cDNA synthesis for Fresh_0307A, Bad_0207B, Bad_0307B | + | Results:<br> |
| + | *Fresh_0207A: 274,3 ng/ul<br> | ||
| + | *Fresh_0207B: 247,2 ng/ul<br> | ||
| + | *Bad_0207A: 232,9 ng/ul<br> | ||
| + | *Bad_0207B: 42,9 ng/ul ------> not enough (treshold = 60 ng/ul).<br> | ||
| + | *Fresh_0307A: 249,8 ng/ul <br> | ||
| + | *Fresh_0307B: 241,0 ng/ul (but low quality)<br> | ||
| + | *Bad_0307A: 249,8 ng/ul<br> | ||
| + | *Bad_0307B: 69,9 ng/ul<br><br> | ||
| + | |||
| + | kept all cDNA in -80 freezer. repeated cDNA synthesis for Fresh_0307A, Bad_0207B, Bad_0307B<br><br> | ||
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| + | NEW POSSIBLE BACKBONE! Also for biobrick submission<br><br> | ||
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| + | Nisa/Emeraldo<br><br> | ||
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| + | 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.<br><br> | ||
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| + | 2.) O/N the E.coli containing the new plasmid(pSAC-CM) for plasmid isolation tomorrow.<br><br> | ||
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| + | 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.<br><br> | ||
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| + | <br> | ||
| + | <A HREF="https://2012.igem.org/Team:Groningen/Notebook"><FONT COLOR=#ff6700>Back to notebook</FONT></A> | ||
| + | </p><br><br> | ||
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| + | {{Template:SponsorsGroningen2012}} | ||
Latest revision as of 16:56, 25 September 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.
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.
Back to notebook
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