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Team:Lyon-INSA-ENS/Realisation/Week10
From 2011.igem.org
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Transformation and plating of NM522 on LB with : p157 (2 µL) + Spc, p115 (2 µL) + Spc, p56 (2 µL) + Amp, p10 (3 µL) + Amp, pIG30 (2 µL) + Cm, p127 (2 µL) + Kn, p116 (5 µL) + Kn <br> <br> | Transformation and plating of NM522 on LB with : p157 (2 µL) + Spc, p115 (2 µL) + Spc, p56 (2 µL) + Amp, p10 (3 µL) + Amp, pIG30 (2 µL) + Cm, p127 (2 µL) + Kn, p116 (5 µL) + Kn <br> <br> | ||
| + | |||
| + | Results of the LB+Amp tests from the previous day : MC4100, NM522+piG4, PHL818, the new NM522 don't grow.<br> | ||
| + | NM522 and NM522 from the collection produce a few colonies : the collection was contaminated by an AmpR strain.<br> | ||
| + | NM522 in the collection (S11) is replaced by the new NM522. <br><br> | ||
| + | |||
| + | Storage of S20. <br> | ||
| + | Plating of S20 on home-made LB+Amp dishes and start of 5mL liquid cultures in LB+Amp. <br><br> | ||
| + | |||
| + | Test of 2 other batches of LB+Amp dishes by plating the new NM522 on them.<br><br> | ||
| + | |||
| + | Plating of NMYO on LB+Kan from an old solid culture.<br><br> | ||
| + | |||
| + | Start of 5mL cultures for 24 well plates in M63G medium, with 50µL antibiotic if appropriate, from 10µL of saturated cultures : PHL818, NM522, NM522+pUC18 (Amp), S18(Amp). | ||
| + | |||
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| + | Result of the test of the 2 batches of LB+Amp : NM522 doesn't grow so they seem efficient.<br><br> | ||
| + | |||
| + | Midiprep of pIG6 (=pUC18)<br> | ||
| + | Digestion by E and electrophoresis to control the plasmid : one band at 2.5-3kb which is coherent. <br> | ||
| + | Nanodrop quantification : c=83ng/µL, 260/280=2.02 <br> | ||
| + | Storage of 300µL of the plasmid at -20°C.<br><br> | ||
| + | |||
| + | Start of a 5mL preculture of NMYO from the previous dish <br><br> | ||
| + | |||
| + | Start of 50mL cultures (500µL antibiotic if necessary and 100µL bacteria ) of NM522 + p10 (Amp),NM522 + p127 (Kan),NM522 + p56 (Amp),NM522 + p157 (Spc),NM522 + p115 (Spc), | ||
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Revision as of 08:14, 29 August 2011
Week 10
From Monday the 22th of August to Friday the 26th of August 2011
Monday
Both NM522 and NM522/pIG24 grew on LB+Amp ( liquid and solid ) : antibiotic concentration in liquid was not sufficient, a more concentrated solution of Amp (10mg/mL which corresponds to 100X) is made and sterilized by filtration.
Creation of our own LB+Amp plates by plating 200µL of Amp on a LB plate.
Plating of several AmpS strains on the previous plates : PHL818, MC4100, NM522+pIG4, NM522 from the collection, NM522 from the previous LB+Amp plate, a new NM522 found in another lab.
Plating of 10µL of the new NM522 on LB
Dissolution of 0.08g of Spectinomycin ( C14H24N2O7 + 2 HCl + 5 H20 ) into 5mL water and filtration to obtain a solution at 10mg/mL.
QIAGen extraction of NM522 and NM522+pIG4 and electrophoresis : no plasmid is in the strains.
Transformation and plating of NM522 on LB with : pUC18 (=pIG6) (3 µL) + Amp, pIG25 (3 µL) + Cm
Transformation and plating of S19 on LB and the antibiotic Amp with : p157 (3 µL) + SpcR, p115 (3 µL) + SpcR, p127 (3 µL) + Kn, p116 (3 µL) + Kn
Tuesday
Transformation and plating of NM522 on LB with : p157 (2 µL) + Spc, p115 (2 µL) + Spc, p56 (2 µL) + Amp, p10 (3 µL) + Amp, pIG30 (2 µL) + Cm, p127 (2 µL) + Kn, p116 (5 µL) + Kn
Results of the LB+Amp tests from the previous day : MC4100, NM522+piG4, PHL818, the new NM522 don't grow.
NM522 and NM522 from the collection produce a few colonies : the collection was contaminated by an AmpR strain.
NM522 in the collection (S11) is replaced by the new NM522.
Storage of S20.
Plating of S20 on home-made LB+Amp dishes and start of 5mL liquid cultures in LB+Amp.
Test of 2 other batches of LB+Amp dishes by plating the new NM522 on them.
Plating of NMYO on LB+Kan from an old solid culture.
Start of 5mL cultures for 24 well plates in M63G medium, with 50µL antibiotic if appropriate, from 10µL of saturated cultures : PHL818, NM522, NM522+pUC18 (Amp), S18(Amp).
Wednesday
Result of the test of the 2 batches of LB+Amp : NM522 doesn't grow so they seem efficient.
Midiprep of pIG6 (=pUC18)
Digestion by E and electrophoresis to control the plasmid : one band at 2.5-3kb which is coherent.
Nanodrop quantification : c=83ng/µL, 260/280=2.02
Storage of 300µL of the plasmid at -20°C.
Start of a 5mL preculture of NMYO from the previous dish
Start of 50mL cultures (500µL antibiotic if necessary and 100µL bacteria ) of NM522 + p10 (Amp),NM522 + p127 (Kan),NM522 + p56 (Amp),NM522 + p157 (Spc),NM522 + p115 (Spc),
Thursday
Friday
