Team:Duke/Notebook/MayJun

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Revision as of 16:12, 29 July 2014

May 29
Objective: Prepare Chemically competent E. Coli
Matthew Faw

Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli

  • Once E. Coli were prepared, the solution was aliquoted into 12 labeled tubes (450l in each tube) and tubes were stored in iGEM box in -80C cooler on far left

Next Steps:

  • Transformation

June 1
Objective: Transform Chemically competent E. Coli (CCEC)
Matthew Faw

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

  • Followed Charlie’s Cloning Protocols
    • Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
  • Plated the transformed DNA and put in incubator at 37C
  • Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

  • Look at plates, compare cultures

June 2
Objective: Redo yesterday’s transformation
Matthew Faw

Due to my failure to correctly conduct the transformation yesterday, it was necessary to redo the transformation.

  • Followed Charlie’s Cloning Protocols
    • Added 1 ul of .5, 5, 10, 20, 50, and 0 (control) pg/lof RFP Construct to chemically competent E. Coli (CCEC), and followed procedure
  • Plated the transformed DNA on 6 separate plates, put in 37C overnight
  • Results: (6/3/14)-Transformation unsuccessful. Try the transformation again with Charlie’s cells and with new cells grown using iGEM’s protocol

Next Steps:

  • Look at plates, compare cultures, etc.
6/4/14 Objective: Prepare buffers and mediums for new CCEC protocol Matthew Farnitano, TJ Ciesla, Mike Zhu, Charlie Cooper Prepare SOB Medium for bacterial transformation Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells Made 1 L, autoclaved and stored in two 500 mL containers in cold room medium still appeared cloudy before autoclave--may just be new recipe Prepare CCMB 80 Buffer for making chemically competent E. coli cells Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells Made 1 L, filtered and stored in two 500 mL containers in cold room pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed Autoclave two 500 mL culture flasks For CCEC protocol With water inside to remove detergent residues Next steps: Prepare CCEC Objective: Attempt to grow some transformed cell cultures with Charlie’s CCEC Matthew Faw The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed. Today, we are trying to see if we can get any transformed cells to grow in plates. Followed Charlie’s Cloning protocols,with slight modifications Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure Plated the transformed DNA on 2 separate plates, put in put in 37C overnight Results: No colonies grew (6/5/14) Next Steps: -Examine plates to see if any cultures grew -Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found here: http://parts.igem.org/Help:Protocols/Competent_Cells -Lab currently in the process of making these CCEC Objective: Prepare pdCas9 marrafini and pCsy4 Arkin plasmids to be miniprepped Matthew Faw, Charlie Cooper Prepare pdCas9 and pCsy4 to be miniprepped tomorrow Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol Put pCsy4 in a culture tube with 5ml SOC+Amp Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them Next steps: Miniprep plasmid DNA

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