ETHZ/labnotes

• Preparing the Solutions

• Prepare competent cells for all parts
• Transformation of all the parts

• Preparing the grown cultures (12) for the MINIPREP
  (o/n cultures)

• MINIPREP of the grown (10) o/n cultures
• Gelelectrophoresis of the grown cultures (step: 0.8% Agarose)

• 7 working parts/plasmids (step after "DIGESTS"):
  (B0034, R0062, R0053, E0434, B0015, R0010, E0422)
• 4 parts/plasmids minipreped:
  (R0040, R0051, Q04121, C0053)

Christos
Markus
Stefan

• Prepare competent cells
• Transformations of J23100, J37033, Q04400, Q04510
• Control Restrictions (step after "MINIPREP")
  R0040, R0051, Q04121, C0053

• o/n culture (E.Coli Top10)
• Control Restrictions (didn't work)

• Start Preparing competent cells (for J23100, J37033, Q04400, Q04510)
  

Evening Shift:

• Transformations of J23100, J37033, Q04400, Q04510

• Prepared competent cells (stored in the -80°C freezer in the basement)
  

Evening Shift:

• Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday)
• Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4%

• Ligation (step: "LINK ASSEMBLY"):
  R0053 + E0422
  R0010 + E0422
  R0010 + E0434
  S/P: R0053, R0010
  X/P: E0422, E0434

• Ligation didn't work due to bad quality of enzymes (probably)

• Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)
• Transformation of #13 and #14

• Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510)
  One batch is miniprepped (after step 19 in the miniprep procedure) and a second batch is frozen as a backup (which is to be miniprepped from step 3 on)
• Transformation of #13 (Q04121) and #14 (C0053)
  Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)

Markus, Christos, (Martin)

• o/n of #13 and #14
• Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful

• #13 and #14 didn't grow
• # 4, 8 and 11 had the plasmid, they were streaked out new on plates, that we have them now on plates
• New white pipette tips prepared (autoclave)
• New bottles of Liquid LB and LB Agar prepared (autoclave)

• Transform pbr322, pcyc177 and pck01

• Transform pbr322, pcyc177 and pck01 and plated them

• Prepare o/n of pbr322, pcyc177, pck01

• o/n of pcyc177, pck01
• the plates of pcyc177 and pck01 are in the fridge
• transformed pbr322 because the culture didn't grow on the plate

• Prepare new competent cells
• Miniprep pcyc177 and pcK01
• prepare new o/n culture of pbr322
• Run agarose gel of Minipreped plasmids

• New competent cells prepared, they are now in the -80° Frezzer in the basement, column #17, dark orange box (we have now 30-35 EDTs of competent cells...)
• Minipreped pcyc177 and pck01 (in the -18° freezer, where the antibiotics are)
• pbr322 didn't grow again, so no o/n could be prepared, but we get a culture from Andy on tuesday
• new o/n of pcyc177 and pck01 prepared (3 Falcons each), because we need to have more plasmids
• 2 boxes of blue pipette tips are in the autoclave
• Stefan ran the agarose gel (?)

• Miniprep pcyc177 and pck01
• cut the prepped plasmids to test if we've got the right ones
• run agarose gel to test the cut and uncut ones
• prepare new o/n of pbr322 (from Andy)

• Miniprep of pcyc177 and pck01 (but not yet tested)
• Prepared 3 o/ns of pbr322 (finally ;-) and each 1 o/n of pcyc177 and pck01, just in case there are problems with the miniprep

• Miniprep of pbr322
• Test-Digest of pcyc177 and pck01 and agarose gel...
• Streak out all three plasmids on new plates, so we have them in reserve

• New Plate of pbr322.
• Minipreps and Agarose Gels will be done tomorrow

• Miniprep of pbr322, pacyc177, pck01
• Test with agarose gel

• Gel of the older plasmids -> plasmid present

• Miniprep of pbr322, pacyc177, pck01

• Plasmids miniprepped

• Miniprep pBR322
• annealing of different MCSs
• Digest of pCK01 with BamHI+AseI
• digest of pACYC177 with BamHI+PstI
• digest of pBR322 with EcoRI+PstI

 all digests o/n

Christian

• Gelextraction of backbones pBR322, pCK01, pACYC digest did NOT work
• 1x ligation of MCS inside backbones o/d, Trafo
• 1x ligation of MCS inside backbones o/n

• plate all 3 plasmids for new minipreps

Christian

• Trafo of o/n ligations
• o/n cultures of putative clones

Christian

• Minipreps of putative clones pCK01-MCS and pBR322-MCS
• control digests of putative clones
• new o/n cultures of the putative clones of o/n ligations

Christian

• separation of control digests of putative clones

*pBR322-MCS (Tet-selection) clone2 positive

Christian

• new digest of pACYC177 with BamHI+PstI o/n

Christian