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Team:Paris Liliane Bettencourt/Notebook/2011/09/21/
From 2011.igem.org
Contents |
Cyrille
TetR-YFP
The Gel extraction was carried on and the tubes pooled. the final concentration is around 10ng/µL
2 times 500ng of the plasmid containing the YFP-tetR wasdigested in E and X 3 times 160ng of the PCR product was digested in E and S
The opened plasmid was PCR purified to eliminate the small fragment. An inactivation of EcoRI enzyme was carried on on the PCR tube at 80°C during 5 min.
In the PCR product, the insert account for 1/3 of the final mass in ng. The digested fragment is 144 bp. The original vector is 3427.
Mvector = 50 ng Cvector = Cinsert = 10 ng/µL
3 ratio will be attempted:
- 1:1 : Minsert = 6,30 ng -> 0,63 µL (ligation in 20 µL)
- 1:6: Minsert = 38 ng -> 3,8 µL (ligation in 20 µL)
- 1:50 Minsert = 315 ng -> 32 µL (ligation in 50 µL)
Then tranformation by eat chock, one hour of growth, and then plating in Cm plates.
Quick change to eliminate PstI inside TetR-YFP
Quick change mutagenesis using the stratagene protocol.
""Dilution""
For te two primers Mother solution 100 µM
-> 1 µL in 20µL -> daughter solution 5µM
125 ng = 8,25 pmol 5 µM = 5.0 pmol/µL
For the Template DNA
Mother concentration: 180 ng/µL
1µL of DNA in 35µL of water
Final concentration: 5µg/µL
""PCR mastermix""
For control tube:
- 5 µL of phusion HF buffer
- 1,65 µL of primer 1 solution
- 1,65 µL of primer 2 solution
- 10µL of template DNA at 5µg/µL
- 30,7 H2O
1µL of phusion
For QCM site:
- 5 µL of phusion HF buffer
- 1,65 µL of primer 1 solution
- 1,65 µL of primer 2 solution
- 1 µL of dNTPs
- 1 - 5 - 10 µL of template DNA at 5µg/µL
- 38,7 - 34,7 - 29,7 µL H2O
1µL of phusion
Primer sequence
GTG GTC GGG GTA GCG GGC GAA GCA TTG GAG GCC GTA GCC GAA GGT GGT CA
Tm primer = 72°C
Habitual temperaturesfor the PCR.
The transformation was done strait away
Danyel
Digestion to check some constructs and for eventual ligations.
S79+S105 digested on E and S, S99+104 digested on E and P, S80 digested on E and P, S98 and S99 on E and S
Characterization of pT7-RBS-GFP-T7ter
The green fluorescence was followed for three different colonies (one negative control) with 6 different concentrations of IPTG on a plate reader for 4hours. These colonies are BL21 strains transformed with S79 (pT7-RBS-GFP-T7ter). The data was analysed and graphs show a clear increase in GFP expression proportional to the increase in IPTG, reaching an eventual limit (around 5mM).
Characterization of tRNA amber suppressor + Pveg-spoVG-GFP amber-TT
pHyperspank-tRNA amber-TT = S99 in pSB1C3 Pveg-spoVG-GFP amber-TT = S136 in pSB1AK3 10 colonies from a double transformation were picked from plates with Amp and Cm. Half of them are fluorescent on the plate, the other half are not. Two cultures were made for each colony, one for the plate reader, the other for a culture with IPTG to be compared with the previous under illumination. (concentrations around 2-4mM). 3 of the colonies showed no fluorescence without IPTG and strong fluorescence with IPTG. The cultures without IPTG of two among these three colonies were chosen and a plate launched for reading (5h). The negative control is a culture of S136 alone and a colony that was not fluorescent on the plate. The concentrations of IPTG are 0mM, 1mM, 2mM, 3mM, 4mM, 5mM. Two rows were prepared for each colony, the second row holding 0.5mM of IPTG more than the previous for all wells. Meanwhile, results are to be compared with the characterization of pHyperspank-RBS-RFP-TT in progress.
