ETHZ/labnotes

From 2007.igem.org

< ETHZ(Difference between revisions)
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(→Week 7)
 
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 +
=== 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.
 +
 +
# [http://partsregistry.org/Part:BBa_B0034 B0034]
 +
# [http://partsregistry.org/Part:BBa_R0062 R0062]
 +
# [http://partsregistry.org/Part:BBa_R0053 R0053]
 +
# [http://partsregistry.org/Part:BBa_J23100 J23100]
 +
# [http://partsregistry.org/Part:BBa_J37033 J37033]
 +
# [http://partsregistry.org/Part:BBa_E0434 E0434]
 +
# [http://partsregistry.org/Part:BBa_B0015 B0015]
 +
# [http://partsregistry.org/Part:BBa_Q04400 Q04400]
 +
# [http://partsregistry.org/Part:BBa_R0010 R0010]
 +
# [http://partsregistry.org/Part:BBa_E0422 E0422]
 +
# [http://partsregistry.org/Part:BBa_R0040 R0040]
 +
# [http://partsregistry.org/Part:BBa_R0051 R0051]
 +
# [http://partsregistry.org/Part:BBa_Q04121 Q04121]
 +
# [http://partsregistry.org/Part:BBa_C0053 C0053]
 +
# [http://partsregistry.org/Part:BBa_Q04510 Q4510]
 +
== Week 1 ==
== Week 1 ==
-
{|
+
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
  ! Date                         
  ! Date                         
  ! TODO's               
  ! TODO's               
Line 8: Line 27:
  |-
  |-
  | Mon, 06. Aug. 2007           
  | Mon, 06. Aug. 2007           
-
|                   
 
-
|                       
 
  |
  |
 +
|                   
 +
* Preparing the Solutions                     
 +
| Sylke, Raphael, Stefan, Markus, Martin, Christos, Joe
  |-
  |-
  | Tue, 07. Aug. 2007           
  | 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           
  | Wed, 08. Aug. 2007           
  |                     
  |                     
-
  |                      
+
  |  
-
  |
+
* Preparing the grown cultures (12) for the MINIPREP <br> (o/n cultures)                     
 +
  | Raphael, Stefan
  |-
  |-
  | Thu, 09. Aug. 2007           
  | 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           
  | Fri, 10. Aug. 2007           
Line 37: Line 62:
  | Sat, 11. Aug. 2007           
  | Sat, 11. Aug. 2007           
  |                     
  |                     
-
  |                      
+
  | no labwork                     
  |
  |
  |-
  |-
-
  | Sun, 12. Aug. 2007        
+
  | Sun, 12. Aug. 2007      
-
|
+
-
* Prepare competent cells                   
+
  |
  |
 +
| labwork cancelled
  |
  |
  |}
  |}
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== Week 2 ==
== Week 2 ==
-
{| class="wikitable"
+
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
! Date                         
! Date                         
! TODO's               
! TODO's               
Line 55: Line 79:
! People
! People
|-
|-
-
| Mon, 13. Aug. 2007        
+
| Mon, 13. Aug. 2007 <br> start at 3 pm       
|
|
 +
* Prepare competent cells
* Transformations of J23100, J37033, Q04400, Q04510
* Transformations of J23100, J37033, Q04400, Q04510
* Control Restrictions (step after "MINIPREP") <br>R0040, R0051, Q04121, C0053
* Control Restrictions (step after "MINIPREP") <br>R0040, R0051, Q04121, C0053
-
* Ligation (step: "LINK ASSEMBLY"): <br> R0053 + E0422 <br> R0010 + E0422 <br> R0010 + E0434 <br> S/P: R0053, R0010 <br> X/P: E0422, E0434                   
+
|                                 
-
|                       
+
* o/n culture (E.Coli Top10)
-
|
+
* Control Restrictions (didn't work)
 +
|  Martin<br> Markus <br> Christos <br> Tim <br>
|-
|-
| Tue, 14. Aug. 2007           
| Tue, 14. Aug. 2007           
-
|                     
+
| Morning Shift: <br>
-
|                      
+
* Start Preparing competent cells (for J23100, J37033, Q04400, Q04510) <br>                      
-
|
+
Evening Shift: <br>
 +
* Transformations of J23100, J37033, Q04400, Q04510
 +
| Morning Shift: <br>
 +
* Prepared competent cells (stored in the -80°C freezer in the basement)    <br>
 +
Evening Shift: <br>
 +
* 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?): <br> Markus, Tim <br> <br> Evening Shift (5pm-...):<br> Martin, Christos
|-
|-
| Wed, 15. Aug. 2007           
| Wed, 15. Aug. 2007           
-
|                    
+
|  
-
|                       
+
* Ligation (step: "LINK ASSEMBLY"): <br> R0053 + E0422 <br> R0010 + E0422 <br> R0010 + E0434 <br> S/P: R0053, R0010 <br> X/P: E0422, E0434
|
|
 +
* Ligation didn't work due to bad quality of enzymes (probably)                       
 +
| From 12:<br> Martin, Markus<br><br>
|-
|-
| Thu, 16. Aug. 2007           
| 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) <br> 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) <br> 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           
| 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           
| Sat, 18. Aug. 2007           
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|
|
|}
|}
 +
 +
== Week 3 ==
 +
 +
Little rearrangements of the parts. Planning of the sequences to order them.
 +
 +
 +
== Week 4 ==
 +
 +
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
 +
! 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 ==
 +
 +
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
 +
! 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 ==
 +
 +
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
 +
! 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 ==
 +
 +
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
 +
! Date                       
 +
! TODO's             
 +
! Completed             
 +
! People
 +
|-
 +
| Mon, 17. Sept. 2007         
 +
|
 +
Hönggerberg:
 +
* new digest of pACYC177 with BamHI+PstI o/n
 +
Zentrum:
 +
* digest of pACYC177, pBR322 AP
 +
* ligation of 177 and 322AP
 +
|
 +
Zentrum:
 +
* 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)
 +
|
 +
Hönngerberg:<br> Christian <br> Zentrum:<br> Martin, Raphael
 +
|-
 +
| Tue, 18. Sept. 2007         
 +
|
 +
Hönggerberg:
 +
* different control digests of pBR322-MCS (Tet) (see last week)
 +
* separation of pACYC177 digest
 +
Zentrum:
 +
* 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
 +
|
 +
Hönggerberg:<br>
 +
> no DNA on pACYC177 digest-gel, only degradation smear<br> <br>
 +
Zentrum:
 +
* 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
 +
  |
 +
Höngg:<br>
 +
Christian <br>
 +
Zentrum:<br>
 +
Martin, Raphael
 +
|-
 +
| Wed, 19. Sept. 2007         
 +
|
 +
Zentrum:
 +
* 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         
 +
|
 +
|
 +
|-
 +
| Thu, 20. Sept. 2007         
 +
|
 +
|
 +
|
 +
|-
 +
| Fri, 21. Sept. 2007         
 +
|                   
 +
|
 +
|
 +
|-
 +
| Sat, 22. Sept. 2007         
 +
|                   
 +
|                       
 +
|
 +
|-
 +
| Sun, 23. Sept. 2007       
 +
|
 +
|
 +
|
 +
|}
 +
 +
== Week 8 ==
 +
 +
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
 +
! 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       
 +
|
 +
|
 +
|
 +
|}

Latest revision as of 19:12, 18 September 2007

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

Hönggerberg:

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

Zentrum:

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

Zentrum:

  • 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)

Hönngerberg:
Christian
Zentrum:
Martin, Raphael

Tue, 18. Sept. 2007

Hönggerberg:

  • different control digests of pBR322-MCS (Tet) (see last week)
  • separation of pACYC177 digest

Zentrum:

  • 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

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

Zentrum:

  • 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

Höngg:
Christian
Zentrum:
Martin, Raphael

Wed, 19. Sept. 2007

Zentrum:

  • 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
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
Retrieved from "http://2007.igem.org/wiki/index.php/ETHZ/labnotes"