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

---

Pouring of Cm+Amp dishes

Preparation of new Spc at 10mg/mL from 0.15g ( C14H24N2O7 + 2 HCl + 5 H20 ) into 10mL water.

Start of 5mL cultures of NM522+p10 in M63G or LB/2.

Friday

---

Digestion of R1, R2, C1, N1, N2 minipreps by E and electrophoresis :

R2 : 2kb, not good
R1 : 2.8kb, validated.
N2 : 2kb, 5kb , not good
N1 : 8kb , not good
C1 : 3kb, 2.5kb , not good
Start of 2X5 mL cultures of NMYO ( 5mL LB, 50µL saturated NMYO, 50µL Kan)

Because no NiCoT plasmid was correct, the transformation was abandonned.