IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/29
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Eric F + Jason G.
Staph OD Growth Curve
Removed index plates from 37C incubator and place in 4C fridge @ 1422
Gel power source borrowed from AMBL because the one in Lagally Lab was not working.
To do for tomorrow:
Vicki Ma 19:28, 29 June 2010 (EDT)
The Plan for the Day
After doing some research we found out that the psB1C3, psB1T3, psB1A3, psB1K3 standard 3A assembly backbones do NOT contain ccdB but actually contain an RFP producing construct. Therefore the transformed cells from last week are not somehow randomly infected with RFP producing DNA, but are actually functioning as they are supposed to. During the previous transformation the resuspended DNA was potentially contaminated by an unautoclaved pipette tip, therefore the DNA was resuspended from the redundant Parts Distribution plate we received.
The overnight cultures of the 4 psB plasmids were picked from selective plates which were the product of a transformation of the potentially contaminated DNA. Since a) The cultures grew on selective plates and b) were visibly red, it is assumed that the cultures contain the proper plasmid. These overnight cultures will be used to make glycerol stocks and mini-preps of the plasmid DNA. Eric Finlay 12:22, 30 June 2010 (EDT)
Testing psB1C3 From Last Year
For most of the experiments so far, we have been using psB1C3 DNA that Hank Yu (on the team last year) made and stored in the 4 degree fridge. I decided to check whether it contained ccdB, so I've transformed it into some DH5(alpha) competent cells and some Db3.1 competent cells. If the DH5(alpha) cells do not grow, and the Db3.1 cells do, it will show that the plasmid backbone contains ccdB. Full procedure (from lab book) will be posted tomorrow when I get back to the lab.
Making chloramphenicol plates
Following the Biobrick 3-A method, prefix parts (promoter + RBS) and suffix parts (fluorescence protein + terminator were joined). The vector used was PSB1A3 (from 2009). The parts joined will be listed below.
Since there are 3 prefix parts and 2 suffix parts, the number of possible combinations is 6, which were all ligated following UBC iGEM standard protocols. The ligation time was 1.5 hours. The combined parts were named 6, 7 , 8, 9 , 10 , 11.
After ligation, the combined parts were transformed into DH5(alpha) competent cells using standard UBC iGEM protocols. The incubation time was 1 hour and 10 μL of ligation mixture was used per 100 μL aliquot of competent cells. The cells were then plated on ampicillin-LB plates.
To do: PCR verify the parts are joined. Send the parts in for sequencing to confirm the parts are present and correctly inserted. Prepare to make electrocompetent Staph aureus cells, following Rafael's protocol.