IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/29

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Staph OD Growth Curve

 * Removed overnight staph culture + B2 glass tube (control) from 37 shaker (10:10am) (JG)
 * Control showed growth
 * Had to re-do experiment and check all components
 * Autoclave the B2 broth
 * Remake the RN4220 glycerol stock (scraping was not done aseptically) Eric Finlay 20:58, 29 June 2010 (EDT)


 * Overnight glass tube with staph aureus culture in 37C shaker; have to remove before 9 am tomorrow (June 30th)

Gel verification

 * Gel verifying the PCR products from last day.
 * Protocol: Gel verification protocol in iGEM training manual. See here

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. DNA: 10uL (no dilution); loading dye: 2uL; Total: 12uL loaded in wells Machine conditions: ~105V, 1x TBE buffer, 1 hour Gel arrangement: Results:
 * band around 1000bp for 6Ta, 8Ta, 11Ta
 * 10Ta had too much agar in the PCR tube and therefore could not be geled

To do for tomorrow: Vicki Ma 19:28, 29 June 2010 (EDT)
 * 1) Do individual colony PCR for 6Ta, 8Ta, 10Ta, 11Ta (from index plates)
 * 2) Gel verification (if enough time)

The Plan for the Day

 * Autoclave B2 (Not Completed)
 * Resuspend psB1A3 (Plate 1, Well 1C), psB1C3 (Plate 1, Well 3A), psB1K3 (Plate 1, Well 5A) and psB1T3 (Plate 1, Well 7A) and dispose of previous resuspensions due to potential contamination (Completed)
 * Begin making Glycerol stocks (start overnight cultures today) of:
 * psB1A3 (Overnight culture: Completed)
 * psB1T3 (Overnight culture: Completed)
 * psB1K3 (Overnight culture: Completed)
 * psB1C3 (Overnight culture: Completed)
 * S. Aureus RN4220 (Overnight culture: Completed)
 * The overnight cultures did not have a control
 * Make Chloro plates (Completed)
 * Transform Hank's psB1C3 into Db3.1 and Dh5α (Completed)

psB Plasmids
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)
 * All four parts (listed below) were resuspended aseptically successully
 * psB1C3
 * psB1K3
 * psB1T3
 * psB1A3
 * Previously resuspended parts were discarded due to potential contamination

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.
 * Transforming Hank's psB1C3 to confirm presence of ccdB
 * Using:
 * 2 Aliquots of competent DH5α
 * 2 Aliquots of competent Db3.1
 * Following the transformation procedure from Common Protocols
 * Competent cells were removed from -80 degree C freezer at 1415
 * Half hour incubation began at 1445
 * Spread 4 LB Agar only plates w/ chloro
 * Began heat shock @ 1515
 * In the incubator @ 1520
 * Removed from incubator at 1635
 * Spread each plate (named same as above) with 450 μL of the transformed cells
 * Let dry next to the flame for 15 minutes, then placed in 37 degree incubator at 1653
 * Must be out by 1053 tomorrow Eric Finlay 12:22, 30 June 2010 (EDT)

Making chloramphenicol plates
(Jason, Melody)
 * Microwaved 1 400ml bottle LB Broth
 * Added 400ul chloramphenicol
 * Used 300 ml and poured 15 plates (stored in 4C freezer)
 * 100 ml unused (solidified before pouring was complete)

Next tasks:
 * Make 400ml LB broth x 2
 * pour Kanamycin and tetracycline plates

Phillip
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.