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Team:Nevada/Notebook/Month2
From 2013.igem.org
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==June 1== | ==June 1== | ||
Jasmine miniprepped the TOP10 cultures and transformed the DNA into LMG149 and plated onto LB AMP plates. | Jasmine miniprepped the TOP10 cultures and transformed the DNA into LMG149 and plated onto LB AMP plates. | ||
Latest revision as of 03:52, 28 September 2013
June 1
Jasmine miniprepped the TOP10 cultures and transformed the DNA into LMG149 and plated onto LB AMP plates. Jasmine worked on the donation website.
June 3
Monday Jasmine did a colony check and cultured two positive colonies each for the Erwinia and Xanthomonas in pBAD (LMG149) both without and without glucose. Chantal transformed 100 microliters of ER+SOC and 100 microliters of ICE+SOC to top 10 cells and plated 100 microliters onto LB CM plates. Jon did extensive internet research pertaining to Ice Nucleating Proteins and cell surface detection methods. Jon contacted plant pathologists at UC Berkeley
June 4
Christian made 3 transformations; GFP, OMPA, and INP fused with YFP into TOP10 cells. GFP and OMPA were successful on AMP plates while INP fused with YFP needed to be re-plated on CM plates. The plasmid stocks of these genes are in our box in the freezer. Christian and Rebecca also made LB-AMP plates and tested them. LB-amp plates in the fridge were also tested. Cody performed a purification of PSB1C3. Cody performed a ligation of PSB1C3 and Erwinia and PSB1C3 and Xanthomonas as well as a relegation of PSB1C3 for control.
June 5
Christian made 5ml liquid cultures (LB-Amp) of the cells with GFP and OMPA. Jon began planning a trip to the Bay Area to meet with plant pathologists at various universities.
June 6
Christian made 5ml liquid culture (LB-CM) of YFP+INP. Christian Made glycerol stocks for pBAD in LMG194 cells for Erwinia (Colony 4, 7) and Xanthamonus Colony 8 (A, B) Jon collected data on the environmental consequences of pesticide use and returned emails to plant pathologists at UC Berkley.
June 5
Christian made 5ml liquid culture (LB-CM) of YFP+INP. Christian Made glycerol stocks for pBAD in LMG194 cells for Erwinia (Colony 4, 7) and Xanthamonus Colony 8 (A, B) Jon collected data on the environmental consequences of pesticide use and returned emails to plant pathologists at UC Berkley.
June 6
Cody, Haley and Chris performed a top 10 transformation of INP/YFP, GFP and OMPA. Cody performed a Gibson assembly of INP/YFP, GFP and OMPA. Jon continued work on general project research and itinerary design.
June10-16
Monday Christian ran the INP+YFP, GFP, and OMPA from last week’s PCR and found that only GFP had a band. Re-did PCR from Friday but modified the protocol. 1 µl of DMSO was added to OMPA and YFP+INP and PCR was run using PHUSION protocol/setting. Jon continued work on general project research and itinerary design. Tuesday Christian ran the DNA from Monday on a gel to confirm the correct band sizes. Christian then did PCR purification of the 3 genes and digested with DPN1 enzyme overnight. Jon continued work on general project research and itinerary design.
Wednesday Christian did PCR purification again on the 3 genes after the DPN1 digestion to remove the enzyme and further concentrate the samples. He ran them on a gel again to confirm the correct identity. He then did a Gibson assembly (YFP+INP alone, and GFP with OMPA) and transformed into cells. Jon continued work on general project research and itinerary design. Thursday Christian did a colony check on the GFP + OMPA and the YFP + INP (x9 colonies). Pseudomonas endolysin genes came in and Christian transformed it into TOP 10 Cells.
Friday Jon continued work on general project research and itinerary design.
June17-23
Monday iGEM Trip
Tuesday iGEM Trip
Wednesday iGEM Trip
Thursday
Friday The team went over the things discussed in the trip and the new directions for the project.
June24–30
Monday Christian made LB-KAN liquid cultures of E. coli containing KZ144 in a pUC57 plasmid (x2).
Tuesday Christian did a miniprep of the two KZ144 E. coli cultures to isolate pUC57 plasmid containing the endolysin. He then ran a PCR to isolate the gene, but the wrong primers were used. A gel was used to analyze the PCR.
Wednesday Christian and Jasmine started making RM media and other buffers. Christian and Jon did a miniprep of GFP, OMPA, and 4 different colonies of OMPA+GFP. They then did a PCR of these genes and ran a gel of the products. Thursday Christian and Jon did a miniprep of the endolysins for Xanthamonus and Erwinia from their original plasmids. Christian and Jon did a PCR purification of Jasmine’s PCR product for her 2 endolysins.
Friday Jon ran an Invitrogen control PCR to trouble shoot some issues which had arisen. Christian grew liquid cultures LB-AMP of the pBAD TOP10 expression control. He also grew 6 liquid cultures of LB-AMP-CM to make sure the X-pBAD BL21 cells have both AMP and CM resistance.
