Team:Paris Liliane Bettencourt/Notebook/2011/09/04/

From 2011.igem.org

Kevin

Biobrick the YFP:tetR fusion protein

Let's start again with good primers to biobrick the protein.

PCR

3 Temperatures of primer annealing: 85 - 58 - 48 °C
Primers : pFX234 FW - pFX234 Reverse CP
And PCR purification after

Gel of PCR products -> Column 1 and 4 = tetR:YFP

Digestion

Digestion in ES for S27 and 2 samples of PCR products. PCR Purification again

Gel of Digested ES PCR products -> Column 1 and 2 = tetR:YFP - Digested ES S27 -> Column 4

It's ok for tetR:YFP digested but we didn't see anything for digested S27.
Do I have made a mistake when I load the gel or the digestion didn't work ? Waiting for transformation to know

Ligation

1h at microscopy room and storage on freezer in "Sevrès box".

To be continued

Transformation, sequencing and glycerol.

Adrien

-80°C PY79 are resistant to TetR up to 75 μg.mL ^-1 which is better than the 150μg.mL ^-1 than the -20°C glycerols of PY79.
Conclusion: to do efficient electro-poration or chemical transformation into Bacillus and using the multi-host vector pHM3 we must use PY79 from the -80°C glycerol.
Tomorrow, Babak will make four glycerol for the -20°C that are going to replace the existing one out of the 4 falcon I launched today.

Cambridge 2008 amylase test was redone with control on the same plate. To be continued by Babak.