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Team:Paris Liliane Bettencourt/Notebook/2011/09/04/
From 2011.igem.org
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Digestion in ES for S27 and 2 samples of PCR products. PCR Purification again | Digestion in ES for S27 and 2 samples of PCR products. PCR Purification again | ||
[[File:geldigyfptetr.jpg|450px|thumb|center|Gel of Digested ES PCR products -> Column 1 and 2 = tetR:YFP - Digested ES S27 -> Column 4]] | [[File:geldigyfptetr.jpg|450px|thumb|center|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.<br> | ||
| + | Do I have made a mistake when I load the gel or the digestion didn't work ? Waiting for transformation to know | ||
====Ligation==== | ====Ligation==== | ||
Revision as of 14:33, 4 September 2011
Contents |
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
Digestion
Digestion in ES for S27 and 2 samples of PCR products. PCR Purification again
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.
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.
