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Team:Duke/Notebook/MayJun
From 2014.igem.org
May 29
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
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
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.
June 4
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
- 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
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:
- Lab currently in the process of making these CCEC
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
