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