From 2014.igem.org
| May 2014 |
| Month 2 of Project |
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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:
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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