Team:Duke/Notebook/MayJun

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

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

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.

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

Results: Plates transformed 6/4 had no colonies (see for results)

Stored glycerol stocks (15% glycerol) of pdCas9 (A1) and pdCas9 (A2) at -80

Miniprep our pdCas9 and pCsy4

• We miniprepped both pdCas9 and pCsy4 following standard protocol included in the miniprep kit. We were unsure of how successful our miniprep of pdCas9 would be because it was low copy.
• We took our own miniprep kit, prepared all solutions necessary, and labeled as ours. Use this miniprep kit from now on.
• We analyzed the concentrations of our plasmids with the spectrophotometer.

Results (6/5/14): Both minipreps were successful, with pdCas9 at concentration 103.4 ng/ulpCsy4 at concentration 138.1ng/ul

• Stored in iGEM 2014 Box 1, A1 and A2

Next steps:

• Wait for oligos ordered 6/4/14 in order to use these plasmids in construction.