ETHZ/labnotes

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Revision as of 18:08, 17 September 2007 by Christian Kemmer (Talk | contribs)

Contents

Part/Plasmid Numberings

The following are the numbering of the parts/plasmids that we use in the lab. Please use the same numberings if you label something when working in the lab. Extend the list for new concatenated parts.

  1. [http://partsregistry.org/Part:BBa_B0034 B0034]
  2. [http://partsregistry.org/Part:BBa_R0062 R0062]
  3. [http://partsregistry.org/Part:BBa_R0053 R0053]
  4. [http://partsregistry.org/Part:BBa_J23100 J23100]
  5. [http://partsregistry.org/Part:BBa_J37033 J37033]
  6. [http://partsregistry.org/Part:BBa_E0434 E0434]
  7. [http://partsregistry.org/Part:BBa_B0015 B0015]
  8. [http://partsregistry.org/Part:BBa_Q04400 Q04400]
  9. [http://partsregistry.org/Part:BBa_R0010 R0010]
  10. [http://partsregistry.org/Part:BBa_E0422 E0422]
  11. [http://partsregistry.org/Part:BBa_R0040 R0040]
  12. [http://partsregistry.org/Part:BBa_R0051 R0051]
  13. [http://partsregistry.org/Part:BBa_Q04121 Q04121]
  14. [http://partsregistry.org/Part:BBa_C0053 C0053]
  15. [http://partsregistry.org/Part:BBa_Q04510 Q4510]

Week 1

Date TODO's Completed People
Mon, 06. Aug. 2007
  • Preparing the Solutions
Sylke, Raphael, Stefan, Markus, Martin, Christos, Joe
Tue, 07. Aug. 2007
  • Prepare competent cells for all parts
  • Transformation of all the parts
Sylke, Raphael, Stefan, Markus, Martin, Christos, Joe
Wed, 08. Aug. 2007
  • Preparing the grown cultures (12) for the MINIPREP
    (o/n cultures)
Raphael, Stefan
Thu, 09. Aug. 2007
  • MINIPREP of the grown (10) o/n cultures
  • Gelelectrophoresis of the grown cultures (step: 0.8% Agarose)
Raphael, Stefan, Martin, Christos, Joe
Fri, 10. Aug. 2007
  • 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

Sat, 11. Aug. 2007 no labwork
Sun, 12. Aug. 2007 labwork cancelled

Week 2

Date TODO's Completed People
Mon, 13. Aug. 2007
start at 3 pm
  • 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)
Martin
Markus
Christos
Tim
Tue, 14. Aug. 2007 Morning Shift:
  • Start Preparing competent cells (for J23100, J37033, Q04400, Q04510)

Evening Shift:

  • Transformations of J23100, J37033, Q04400, Q04510
Morning Shift:
  • 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%
Morning Shift (9am-1pm?):
Markus, Tim

Evening Shift (5pm-...):
Martin, Christos
Wed, 15. Aug. 2007
  • 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)
From 12:
Martin, Markus

Thu, 16. Aug. 2007
  • 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)

Fri, 17. Aug. 2007
  • 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)
Martin
Sat, 18. Aug. 2007
Sun, 19. Aug. 2007

Week 3

Little rearrangements of the parts. Planning of the sequences to order them.


Week 4

Date TODO's Completed People
Mon, 27. Aug. 2007
Tue, 28. Aug. 2007
Wed, 29. Aug. 2007
Thu, 30. Aug. 2007
Fri, 31. Aug. 2007
Sat, 01. Sept. 2007
  • Transform pbr322, pcyc177 and pck01
  • Transform pbr322, pcyc177 and pck01 and plated them
Stefan
Sun, 02. Sept. 2007
  • 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
Stefan

Week 5

Date TODO's Completed People
Mon, 03. Sept. 2007
  • 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 (?)
Martin, Stefan
Tue, 04. Sept. 2007
  • 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
Martin, Christian
Wed, 05. Sept. 2007
  • 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
Martin
Thu, 06. Sept. 2007
  • Miniprep of pbr322, pacyc177, pck01
  • Test with agarose gel
  • Gel of the older plasmids -> plasmid present
Christian
Fri, 07. Sept. 2007
  • Miniprep of pbr322, pacyc177, pck01
  • Plasmids miniprepped
Martin
Sat, 08. Sept. 2007
Sun, 09. Sept. 2007

Week 6

Date TODO's Completed People
Mon, 10. Sept. 2007
  • 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

Tue, 11. Sept. 2007
  • 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

Wed, 12. Sept. 2007
  • Trafo of o/n ligations
  • o/n cultures of putative clones

Christian

Thu, 13. Sept. 2007
  • 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

Fri, 14. Sept. 2007
  • separation of control digests of putative clones

*pBR322-MCS (Tet-selection) clone2 positive

Christian

Sat, 15. Sept. 2007
Sun, 16. Sept. 2007

Week 7

Date TODO's Completed People
Mon, 17. Sept. 2007
  • new digest of pACYC177 with BamHI+PstI o/n

Christian

Tue, 18. Sept. 2007
Wed, 19. Sept. 2007
Thu, 20. Sept. 2007
Fri, 21. Sept. 2007
Sat, 22. Sept. 2007
Sun, 23. Sept. 2007

Week 8

Date TODO's Completed People
Mon, 24. Sept. 2007
Tue, 25. Sept. 2007
Wed, 26. Sept. 2007
Thu, 27. Sept. 2007
Fri, 28. Sept. 2007
Sat, 29. Sept. 2007
Sun, 30. Sept. 2007