ETHZ/Biology/Labbook

• 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
• digest of pACYC177, pBR322 AP
• ligation of 177 and 322AP

• digest of pBR322 AP (the concentration of DNA was too low for pacyc177...)
• ligation of pBR322 o/n
• 100 ml o/n culture to MAXIprep pacyc177
• Transformation of pBR322 AP to have it on plates (because andy only miniprepped them)

Christian
Martin, Raphael

• different control digests of pBR322-MCS (Tet) (see last week)
• separation of pACYC177 digest
• Test Digests of pck01 with XbaI, SpeI, PstI, Xba/Pst, Xba/Spe (because all of them should be in the plasmid due to the sequence, and if they are it would be crap!!!)
• Transformation of the ligated pbr322 AP (MCS)
• Prep pacyc177
• Digest prepped pacyc177

> no DNA on pACYC177 digest-gel, only degradation smear

• Plates of pbr322 AP grew
• No Digest of pck01 worked due to too low DNA concentration... (che cazzo di low copy plasmids !!!!)
• Miniprepped only 20 ml of the pacyc o/n culture with Quiagen Kit, the results were great! We have loads of DNA! (thank god! )
• Digest of pacyc177 with BamHI (45 µl), then precipitated, in the gel was still very much DNA, but there were still 3 bands, so we guess, that it hasn't cut, maybe because the BamHI in the center is very old, perhaps we should Digest it in Höngg again.
• Digest of pacyc177 with PstI o/n (pray that it will work!)
• New o/n cultures of pck01 (to prep it like pacyc177), pbr322 AP (to prep it too, to have something on stock again, if the ligation didn't work), top10 (to make new competent cells)
• test digest of pck01 with notI, but due to the low DNA concentration I don't think it will work. I took glooves, if it now work, then we have caught some DNases in the earlier test digests

Christian
Martin
Raphael

• o/n culture of pbr322 AP (MCS), then test digest and see if it is ligated
• Prep of pck01 and test digests (xba, pst, spe, pvuI, notI)
• check the digests of pacyc177 (pst) and pck01 (notI)
• design new linkers for pck01, design primers for PCR for the extraction of SpeI from pck01