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Team:Paris/16 August 2009
From 2009.igem.org
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Lab work
Yesterday transformations results
- g3p ON ligation at 16°C
- Negative control concentrated = 0 bacteria
- 1:1 concentrated = 1 bacteria
- 1:7 concentrated = 1 bacteria
- ClyA ON ligation at 16°C
- Negative control concentrated = 0 bacteria
- 1:1 rapport = 0 bacteria
- 1:7 rapport = 0 bacteria
- 1:1 concentrated = 10 bacteria
- 1:7 concentrated = 0 bacteria
Miniprep
- For the 6 positives clone of pFecA
Glycerol Stock
- For clone n°1 and n°2
- 333ul of Glycerol 60%
- 666ul of ON culture
- Mix and store at -80°C
Digestion
- Digestion for the 6 clones of pFecA ligation, by XbaI and PstI
- Mix
- DNA (Miniprep) : 5ul
- XbaI : 1ul (I think it's too much...but I think to this after doing it)
- PstI : 1ul
- BSA 100x : 0,2ul
- Buffer NEB2 10x : 2ul
- H2O : 9ul
- 2h at 37°C
Gel migration
- 1,5% agarose gel
- 5ul drop
- Good profil, expected band at 130bp. Maybe not enough digest bu we clearly see the 130bp band corresponding to the insert (pFecA). The 2000bp band correspond to pSB1A3
ON culture
- DH5α in order to done more competent bacteria (the stock is shrinking)
- 4 g3p clones (7ml + 7ul Amp)
PCR screening
- Same protocol as yesterday
- Screening of 4 clones (maybe I invert the n°2 and the n°4)
Gel migration
- g3p is around 1450bp... Second confirmation tomorrow but it seems very good!
