Team:Michigan/Pili Expression
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*headed over to the ERB and made two cultures of 5mL LB-Amp (in 50mL conical tubes)--> placed in the Lin Lab 4C fridge | *headed over to the ERB and made two cultures of 5mL LB-Amp (in 50mL conical tubes)--> placed in the Lin Lab 4C fridge | ||
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+ | ==8/17/2010== | ||
+ | '''Kevin, Marc, Alena''' | ||
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+ | Marc inoculated the two cultures made last night (8/16/2010) with pBAD and grew in 37C shaker (~9am to ~6p, 9hrs) | ||
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+ | NOTE REGARDING LB CULTURE MADE ON 8/16/2010 | ||
+ | -Marc noticed the 400mL LB media made in the ERB to be contaminated. | ||
+ | -Our two cultures could have been contaminated but pBAD grew pretty well (saturated) so we're not too worried | ||
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+ | fimE and fimB knock out (K.O.) strains came in | ||
+ | *Marc adds 50uL of kanamycin onto each LB-agar plate and spread evenly with sterile glass beads | ||
+ | **LB-agar plate ~25mL | ||
+ | **Jeremy Minty's advice: more Kan is better than too little --> Kan 100 instead of Kan 50 | ||
+ | **allow the plate to absorb the kanamycin for 2 hours before applying the strains | ||
+ | *Alena follows Jeremy Minty's example of carefully taking out the small filter circle out of the foil with sterile techniques onto a LB-agar plate (previously labeled) and streaked the filter paper w/ inoculating streakers (used 3 of them) | ||
+ | *incubate plates w/ filter paper still inside upside (agar side up) at 37C overnight | ||
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+ | Followed 8/14/2010 protocol (above) for: | ||
+ | *Miniprep for pBAD plasmid | ||
+ | **Last time we used the incorrect spin column (QIAquick spin column; purple; used for PCR purification). This time we used QIAprep spin column (blue; had no cap on the column) | ||
+ | *PCR purification for fimB | ||
+ | **used PCR products labeled C and D tubes | ||
+ | *Digest fimB and pBAD | ||
+ | **This time, made sure the time on the program was set to 12:00:00 | ||
+ | **5 rxns for each fimB and pBAD (10 total tubes, 20uL total volume in each tube) | ||
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Revision as of 01:25, 18 August 2010