Team:Groningen/Labwork/21 August 2013

Sander

made 8 inoculations of j04450 strain in LB-Broth. made inoculations of CheY knockout and wild type bacillus on 0.3% and 0.4% agar with LB nutrient.

Inne

Inoculated the 0.3% and 0.4% agar plates with wt and delta cheY colonies from the plates from 19-08 (same as 0.7% plates). Each strain was grown in duplo. Plates were left to grow overnight in the 37C incubator.

Claudio

The synthetic gene sequences, which were ordered, are 16 and I'll refer to that as S# (see LINK).

S1, S2, S5, S6 and pSB1C3 were digested (NEB enzymes) with EcoRI and PstI.
S16 was digested (FastDigest) with EcoRI and BamHI.
S3 and S4 were digested (FastDigest) with BamHI and PstI.

S1, S2, S5, S6, SS1 (S16 + S3) and SS2 (S16 + S4) were ligated into pSB1C3.
The ligation reactions were transformed into E. Coli DH5α and plated on LB + Cm.

Sebas

The new transformation showed no colonies. The inoculated colonies form the first transformation grew so did a
miniprep. Digested 500ng of plasmid with XbaI and PstI for 1h at 37degrees. No digested DNA was visible on gel
nor for the ondigested sample.
The colonies on the plate were a contamination.

Did a new ligation of GFPdsm (XbaI*PstI) + hy_spnk_munich (XbaI*PstI) in a molar ratio of 1:3 (vector:insert).
Incubated for 1h at 25degrees in shaking waterbath and transformed to E. coli. Incubated plates at 37degrees.