User:Perry/Spring 2007
2/12/07
dissolved 500mg ampicillin salt into 10ml water to prepare 50mg/ml ampicillin stock; aliquotted 1ml into tubes, left in -20dC.
overnight 1ml LB-amp cultures of JLO159, W, H, S, ~4:30pm.
2/13/07
took overnight cultures out ~7:30pm. diluted 1:10, measured OD600.
159- .335, W- .245, H- .231, S- .279
made liquid cultures of 5ml LB-amp + 250ul overnight cultures. incubated 3h, added 5mM IPTG (53ul 0.5M), incubated 2h, diluted 1:10, measured OD600.
159- .106, W- .044, H- .023, S- .057
Aliquotted 1.5mL of H into each tube and proportional volumes of the rest. aliquotted volumes into tubes. centrifuged 8000rpm, 3min. aspirated. reconstituted in 10ul streptavidin aptamer, 10ul B/F hybrid, or 10ul B0/F hybrid.
4uM streptavidin aptamer: 48ul PBS + 2ul streptavidin aptamer (100uM)
4uM B/F hybrid: 46ul PBS + 2ul Bside oligo (100uM) + 2ul Iside oligo (100uM)
4uM B0/F hybrid: 46ul PBS + 2ul DispI oligo (100uM0 + 2ul Iside oligo (100uM)
Wrapped tubes in a napkin, rotated on rotisserie at room temperature for 1h. washed in PBS 3X, reconstituted in 50ul PBS, loaded into clear 96-well plate.
2/28/07
I'm going to shift the project away from streptavidin for now, until I learn more about membrane fractionation or until I get a full streptavidin clone from Takeshi Sano. I'm going towards expressing PDZ repeats on the surface.
Prepared 150ml cultures of JLO66 and JLO159.
3/1/07
Hi-Speed Midiprep of JLO66 and JLO159.
3/2/07
Received primers PDZ1F, PDZ2F, PDZ1RS, PDZ2RS. Dissolved in (10x#nmol)ul for 100uM. Diluted 1:10 for 10uM used in PCR.
Received F11/Na template from Alain; nanodropped at ~10ng/ul. Made a 1:50 dilution.
Prepared six 50ul PCR reactions, adding the 1ul of each of the following to 47ul Platinum PCR Supermix.
PDZ1: F11, PDZ1F, PDZ1RS
PDZ2: F11, PDZ2F, PDZ2RS
PDZ1+2: F11, PDZ1F, PDZ2RS
PDZ1/2/1+2 (1:50): same, using 1:50 F11.
Incubated 95dC 10min, (95dC 30sec, 55dC 30sec, 72dC 60sec)x30, 72dC 10min.
Walker ran 10ul of each PCR reaction on an e-gel.
Lane 1: 1kb+
Lane 5: PDZ1
Lane 6: PDZ2
Lane 7: PDZ1+2
Lane 8: PDZ1 (1:50)
Lane : PDZ2 (1:50)
Lane 10: PDZ1+2 (1:50)
