Team:Heidelberg/Notebook/Killing I/Notebook/week12

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Week 12

Contents

Monday, 10/20/08

Proceedings of phage cloning strategy one

  • the fully mutated insert in pBluescript SK II was digested with XbaI/XhoI and the insert extracted from the gel
  • lambda DNA was also cut with XbaI/XhoI and the backbone also extracted from the gel.
  • the insert was ligated in the lambda backbone over night using standard ligation methods


Phage cloning strategy two

  • Digestion with XbaI/XhoI
    • should be: 3945, 2898
  • Gel

Hd-phage-08-10-20 pBlue insert new XbaI-XhoI.jpg

  • lane 1-8: new minipreps
  • lane 9: old insert fully mutated
  • results: the new minipreps seem to be the old unmutated pBluescript

Tuesday, 10/21/08

Proceedings of phage cloning strategy one

  • the emerging plasmid consisting of our insert in tha lambda phage was transformed in E. coli by chemical transformation and in vitro packaging
  • the bacterias were plated on chloramphenicol and incubated at 28 °C to ensure that the thermolabile cI (cI857) was not degraded


Phage cloning strategy two

  • do again a digestion of mutagenesis pcr minipreps from 10/07/08 but with an additional restriction enzyme (EcoRI)to get a better separation on gel to cut out the backbone
  • digestion with KpnI/AgeI/EcoRI
    • mutagenesis pcr succesful: 300bp, 1.4kb 5.1kb
    • mutagenesis pcr not succesful: 300bp, 1.4kb, 1.7kb, 3.4kb

Hd-phage-pBlue mutkpn.jpg

  • -->mutagenesis pcr on the first three lanes successful
    • cut out 5.1kb band and gel purification kit

Hd-phage-cloningtwo-ligationfragments.jpg

Wednesday, 10/22/08

Proceedings of phage cloning strategy one

  • first small colonies can be seen on chloramphenicol plates.
  • colonies were picked and transferred in liquid media containing chloramphenicol


Thuresday, 10/23/08

Proceedings of phage cloning strategy one

  • colonies could grow in chloramphenicol containing medium --> samll aliquots were transferred in ampicillin containing medium to check for the resisitance
  • colony PCRs were conducted to ensure that our insert is embedded in the growing cells
  • pUB307 was transferred in cells harboring our modified lambda phage by conjugation --> plating on Cm + Kan


Friday, 10/24/08

Proceedings of phage cloning strategy one

  • colonies grew on plates containing chloramphenicol and kanamycin --> were picked and liquid medium inoculated
  • conjugation of our lambda phage into cells harboring T9002 in pSB1A3 --> brought out on plates containing chloramphenicol and ampicillin


Saturday, 10/25/08

Proceedings of phage cloning strategy one

  • we got colonies on the plates with ampicillin and chloramphenicol only when our conjugated cultures were brought out, but not if T9002 or the cells harboring pUB307 and our phage were brought out separately
  • this shows that our phage can be transported by conjugation


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