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 250μL Buffer P1
   - 250μL 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)
   - 350μL 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
   - 50μL water added to center of column
      (let column stand for 1 min)
   - centrifuged for 1 min
   - nanodropped

Digestion

1. Materials

   8μL DNA (R0010, E0241)
   2.5μL 10x BSA
   2.5μL 10x Buffer (Buffer 2)
   11μL dH2O
   0.5μL enzyme 1 and 2 (SpeI and PstI, XbaI and PstI)
   total vol = 25μL

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 0.1μL (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