IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/09/12: Difference between revisions
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- Single digest : with EcoRI and XbaI, add SDW and DNA (according to the previous table, 300mg of DNA), 2μL of fast digest buffer, and 1μL of enzyme | - Single digest : with EcoRI and XbaI, add SDW and DNA (according to the previous table, 300mg of DNA), 2μL of fast digest buffer, and 1μL of enzyme | ||
- | - Gel1 | ||
*Lane2 : HyperladderI | |||
*Lane3 : RBS W1 cut with EcoRI | |||
*Lane4 : RBS W1 cut with XbaI | |||
*Lane5 : RBS W3 cut with EcoRI | |||
*Lane6 : RBS W3 cut with XbaI | |||
*Lane7 : RBS W7 cut with EcoRI | |||
*Lane8 : RBS W7 cut with XbaI | |||
*Lane9 : RBS W9 cut with EcoRI | |||
*Lane10 : RBS W9 cut with XbaI | |||
*Lane11 : RBS W9 uncut | |||
*Lane12 : supercoiled ladder | |||
- Gel2 | |||
*Lane2 : HyperladderI | |||
*Lane3 : RBS S4 cut with EcoRI | |||
*Lane4 : RBS S4 cut with XbaI | |||
*Lane5 : RBS S6 cut with EcoRI | |||
*Lane6 : RBS S6 cut with XbaI | |||
*Lane7 : RBS S8 cut with EcoRI | |||
*Lane8 : RBS S8 cut with XbaI | |||
*Lane9 : RBS S11 cut with EcoRI | |||
*Lane10 : RBS S11 cut with XbaI | |||
*Lane11 : RBS S4 uncut | |||
*Lane12 : supercoiled ladder | |||
*Result | |||
[[Image:gel21092pcr.gif|250px|center]] | |||
The size of our plasmid is about 3000bp (ok on the gel) | |||
*RBS W | |||
- W1 with EcoRI : cut | |||
- W1 with XbaI : uncut, self ligation | |||
- W3 with EcoRI : cut | |||
- W3 with XbaI : uncut, self ligation | |||
- W7 with EcoRI : impossible to see | |||
- W7 with XbaI : uncut, self ligation | |||
- W9 with EcoRI : cut | |||
- W9 with XbaI : uncut, self ligation | |||
*RBS S | |||
- S4 with EcoRI : cut | |||
- S4 with XbaI : uncut, self ligation | |||
- S6 with EcoRI : cut | |||
- S6 with XbaI : 2 band, this plasmid is partially cut!!! Transformation with RBS S | |||
- S8 with EcoRI : cut | |||
- S8 with XbaI : uncut, self ligation | |||
- S11 with EcoRI : cut | |||
- S11 with XbaI : uncut, self ligation | |||
==Double check of RBS S6 and stock== | |||
- Grow RBS S6 in LB with Cm35 | |||
==Check more RBS W== | |||
- Grow RBS W2, 4, 6, 8, 10 in 10mL of LB with antibiotic | |||
==Check PCR products== | ==Check PCR products== | ||
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*Lane11 : RBS W | *Lane11 : RBS W | ||
*Lane12 : HyperladderI | *Lane12 : HyperladderI | ||
*Results | |||
[[Image:gel21091pcr.gif|200px|center]] | [[Image:gel21091pcr.gif|200px|center]] | ||
- agr B : 2 bands? | |||
- agrC : ok | |||
- rep : small band but good size | |||
- backbones : ok | |||
- promoters : ok | |||
- RBS : nothing | |||
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Revision as of 02:46, 16 September 2008
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RBS screening (single digest)- Plasmid miniprep 6 colonies of RBS S and 6 colonies of RBS W (without endo wash buffer) - Nanodrop
- Single digest : with EcoRI and XbaI, add SDW and DNA (according to the previous table, 300mg of DNA), 2μL of fast digest buffer, and 1μL of enzyme - Gel1
The size of our plasmid is about 3000bp (ok on the gel)
- W1 with EcoRI : cut - W1 with XbaI : uncut, self ligation - W3 with EcoRI : cut - W3 with XbaI : uncut, self ligation - W7 with EcoRI : impossible to see - W7 with XbaI : uncut, self ligation - W9 with EcoRI : cut - W9 with XbaI : uncut, self ligation
- S4 with EcoRI : cut - S4 with XbaI : uncut, self ligation - S6 with EcoRI : cut - S6 with XbaI : 2 band, this plasmid is partially cut!!! Transformation with RBS S - S8 with EcoRI : cut - S8 with XbaI : uncut, self ligation - S11 with EcoRI : cut - S11 with XbaI : uncut, self ligation Double check of RBS S6 and stock- Grow RBS S6 in LB with Cm35 Check more RBS W- Grow RBS W2, 4, 6, 8, 10 in 10mL of LB with antibiotic Check PCR products- Gel (2μL of each product)
- agr B : 2 bands? - agrC : ok - rep : small band but good size - backbones : ok - promoters : ok - RBS : nothing |