Team:Evry/Notebook/w8
From 2013.igem.org
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- | We performed | + | We performed 6 golden gates, 3 for the second construction, 2 for the third construction and one for the controle plasmid. |
- | + | We prepared a mix for the 6+1 tubes : | |
<ul> | <ul> | ||
- | <li> 8,89 µl of 1A3 plasmide | + | <li> 7x 1,27 = 8,89 µl of 1A3 plasmide |
- | <li> 12,18 µL of terminator | + | <li> 7x 1,74 = 12,18 µL of terminator |
- | <li> | + | <li> 7x 1,5 = 10,5 µL of T4 Buffer |
- | <li> 3,5 µL of BSA | + | <li> 7 x 0,5 = 3,5 µL of BSA |
- | <li> 3,5 µL of T4 ligase | + | <li> 7 x 0,5 = 3,5 µL of T4 ligase |
</ul> | </ul> | ||
</p> | </p> | ||
<p> | <p> | ||
+ | We then added:<br> | ||
+ | |||
+ | Tube 1. = | ||
+ | <ul> | ||
+ | <li> 0,76 µl of Andersen's promoter | ||
+ | <li> 0,76 µL of RBS | ||
+ | <li> 0,76 µl OF Fur BS | ||
+ | <li> 2,93 µL of sfGFP | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | Tube 2. = | ||
+ | <ul> | ||
+ | <li> 0,76 µl of Andersen's promoter | ||
+ | <li> 0,76 µL of RBS | ||
+ | <li> 0,76 µl OF Fur BS | ||
+ | <li> 2,93 µL of sfGFP | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | Tube 3. = | ||
+ | <ul> | ||
+ | <li> 0,76 µl of Andersen's promoter | ||
+ | <li> 0,76 µL of RBS | ||
+ | <li> 0,76 µl OF Fur BS | ||
+ | <li> 2,93 µL of sfGFP | ||
+ | <li> 4,28 µL of water | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | Tube 4. = | ||
+ | <ul> | ||
+ | <li> 1,25 µl of pLac O | ||
+ | <li> µL of RBS-sfGFP | ||
+ | <li> 3,4 µL of water | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | Tube 5. = | ||
+ | <ul> | ||
+ | <li> 1,25 µl of pLac O | ||
+ | <li> 2,05 µl of EntA | ||
+ | <li> 3,37 µL of EntD | ||
+ | <li> 2,07 µl of EntF | ||
+ | <li> 0,5 µL of water | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | Tube 6. = Controle Plasmid | ||
+ | <ul> | ||
+ | <li> 0,76 µl of Andersen's promoter | ||
+ | <li> 0,76 µL of RBS | ||
+ | <li> 2,93 µl of sfGFP | ||
+ | <li> 4,28 µL of water | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | On the afternoon, we launch another golden gate for EntB/C/E | ||
+ | |||
+ | Tube 7. = | ||
+ | <ul> | ||
+ | <li> 1,27 µl of plasmid 1A3 | ||
+ | <li> 1,5 µL of pLacO | ||
+ | <li> 0,49 µl of EntB | ||
+ | <li> 0,65 µL of EntC | ||
+ | <li> 1,74 µl of terminator | ||
+ | <li> 1,5 µL of Buffer T4 ligase | ||
+ | <li> 0,5 µl of T4 ligase | ||
+ | <li> 0,5 µL of Bsa 1 | ||
+ | <li> 5,88 µL of water | ||
+ | </ul> | ||
+ | </p> | ||
</br> | </br> | ||
<p> | <p> |
Revision as of 14:27, 7 August 2013
Week 8: 5th August - 11th August
Plasmide 3:
We started the week by doing the golden gates over again, meaning the GG 3 and GG 2 (2.1 with FurBS 1, 2.2 with FurBS2 and 2.3 with FurBS3). In fact, our previous control positive had some unwanted white spots (01/08/13), thus suggesting some contaminations during the transformation step. curretnmy waiting
We performed 6 golden gates, 3 for the second construction, 2 for the third construction and one for the controle plasmid. We prepared a mix for the 6+1 tubes :
- 7x 1,27 = 8,89 µl of 1A3 plasmide
- 7x 1,74 = 12,18 µL of terminator
- 7x 1,5 = 10,5 µL of T4 Buffer
- 7 x 0,5 = 3,5 µL of BSA
- 7 x 0,5 = 3,5 µL of T4 ligase
We then added:
Tube 1. =
- 0,76 µl of Andersen's promoter
- 0,76 µL of RBS
- 0,76 µl OF Fur BS
- 2,93 µL of sfGFP
- 0,76 µl of Andersen's promoter
- 0,76 µL of RBS
- 0,76 µl OF Fur BS
- 2,93 µL of sfGFP
- 0,76 µl of Andersen's promoter
- 0,76 µL of RBS
- 0,76 µl OF Fur BS
- 2,93 µL of sfGFP
- 4,28 µL of water
- 1,25 µl of pLac O
- µL of RBS-sfGFP
- 3,4 µL of water
- 1,25 µl of pLac O
- 2,05 µl of EntA
- 3,37 µL of EntD
- 2,07 µl of EntF
- 0,5 µL of water
- 0,76 µl of Andersen's promoter
- 0,76 µL of RBS
- 2,93 µl of sfGFP
- 4,28 µL of water
- 1,27 µl of plasmid 1A3
- 1,5 µL of pLacO
- 0,49 µl of EntB
- 0,65 µL of EntC
- 1,74 µl of terminator
- 1,5 µL of Buffer T4 ligase
- 0,5 µl of T4 ligase
- 0,5 µL of Bsa 1
- 5,88 µL of water
Additionnally, we did the optimization of our PCR over with the temperature gradient for the annealing step. current waiting