IGEM:Harvard/2006/vlau/Notebook/2006-6-14

Miniprep
1. Plasmids (2 samples each) R0010: lac operon promoter E7104: T7 promoter + GFP E0241: GFP 2. Protocol - w/ 5mL samples, 1mL saved for glycerol stock - rest of samples transferred to 1.5 eppendorfs and centrifuged to form pellet - supernatant removed and pellet resuspended in 250 Buffer P1   - 250 Buffer P2 added and tube inverted 4-6 times for mixing (no vortexing to prevent shearing of genomic DNA; let sit for no longer than 5 min) - 350 Buffer N3 added and tube inverted 4-6 times for mixing - centrifuged @ 13,000rpm for 10 min (formation of white pellet) - supernatant transferred to QIAprep spin column and centrifuged for 1 min - 0.5mL Buffer PB added and centrifuged for 1 min (optional) - added 0.75mL Buffer PE for washing and centrifuged for 1 min - flowthrough discarded and centrifuged for 1 min to remove residual buffer - QIAprep column transferred to 1.5 eppendorfs - 50 water added to center of column (let column stand for 1 min) - centrifuged for 1 min - nanodropped

Digestion
1. Materials 8 DNA (R0010, E0241) 2.5 10x BSA 2.5 10x Buffer (Buffer 2) 11 dH2O 0.5 enzyme 1 and 2 (SpeI and PstI, XbaI and PstI) total vol = 25 2. Protocol - checked www.neb.com to determine correct Buffer - digested @ 37dC for 1 hr   - heat shocked @ 97dc for 15 min to inactivate restriction enzymes - added 0.1 (1 unit) of 10,000units/mL CIP to vector DNA (R0010) - incubated vector DNA @ 37dC for 1 hr

Gel Electrophoresis and Purification
1. 1.0% Agarose Gel L. 1: 1 kb Ladder L. 2: R0010 Sample 1 L. 3: R0010 Sample 2 L. 4: E0241 Sample 1 L. 5: E0241 Sample 2