User:Vlau

Contact Info.

vlau@fas.harvard.edu/vhtlau@gmail.com

iGEM:Harvard/2006

Week 1

06.12.06: Mon.

Folding DNA Nanostructures

1. Working Stocks

  44 nM scaffold (20 microL)
  0.99 microM of each oligo

2. Protocol

   - goal: 10 nM scaffold w/ 100 nM each oligo, total rxn vol of 20 microL
   - calculations:
      scaffold = (10 nM)/(44 nM) * 20 microL = 4.5 microL
      oligos = (100 nM)/(990 nM) * 20 microL = 2 microL
   - reaction mixture:
      4.5 microL p7308 scaffold
      2 microL oligos
      2 microL 10x folding buffer (500 mM HEPES ph 7.5, 500 mM NaCl, 100 mM MgCl2)
      11.5 microL dH2O
   - annealing times:
      90 dC, 5'
      65 dC, 20'
      55 dC, 20'
      45 dC, 20'
      37 dC, 30'
   - neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water

Transformation

1. Plasmids

   R0010: lac operon promoter
   E7104: T7 promoter + GFP
   E0241: GFP

2. Protocol

   - let chemically competent OneShot Top10 cells thaw on ice
   - added appropriate DNA to cells and tapped gently to mix
   - let sit on ice for 20 min
   - heat shocked @ 42 dC for 30"
   - let cool on ice for 2 min
   - added 200 microL SOC media
   - shook @ 37 dC for 1 hr
   - pipetted and spread onto agar plates treated w/ ?
   - incubated @ 37 dC overnight agar side-up

06.13.06: Tues.

Gel Electrophoresis w/ 2% Gel

1. Protocol

   - mixed 2 g Ultrapure agarose w/ 100 mL 1X TBE in plastic flask
   - heated for 1.5 min in microwave w/ top loosely screwed on
   - added 3 microL EtBr
   - poured into gel frame and let set for 20 min
   - placed into running box and submerged w/ TBE
   - wells loaded
   - ran gel @ 130 V, 45 min

2. Samples

   L. 1: 1 kB ladder
   L. 2 and 3: DNA nanostructure
   L. 4 and 5: neg control w/ no oligonucleotides
   L. 6 and 7: neg control w/ no scaffold
   L. 8: 1 kB ladder
   loading dye: BTB dye 50% glycerol + 10X TBE
   ladder: 10 microL + 1 microL dye
   samples: 10 microL + 1 microL dye

3. Image

Transformation P. 2

1. Protocol

   - prepared 2 liquid cultures for each transformation
   - added 50 microL of 5 mg/mL amp to 5 mL LB per culture
   - shook @ 180 rpm, 37 dC overnight

06.14.06: Wed.

Miniprep

1. Plasmids (2 samples each)

   R0010: lac operon promoter
   E7104: T7 promoter + GFP
   E0241: GFP

2. Protocol

   - w/ 5 mL samples, 1 mL saved for glycerol stock
   - rest of samples transferred to 1.5 eppendorfs and centrifuged to form pellet
   - supernatant removed and pellet resuspended in 250 microL Buffer P1
   - 250 microL 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 microL Buffer N3 added and tube inverted 4-6 times for mixing
   - centrifuged @ 13,000 rpm for 10 min
      (formation of white pellet)
   - supernatant transferred to QIAprep spin column and centrifuged for 1 min
   - 0.5 mL Buffer PB added and centrifuged for 1 min
      (optional)
   - added 0.75 mL 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 microL water added to center of column
      (let column stand for 1 min)
   - centrifuged for 1 min
   - nanodropped

Digestion

1. Materials

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

2. Protocol

   - checked www.neb.com to determine correct Buffer
   - digested @ 37 dC for 1 hr
   - heat shocked @ 97 dc for 15 min to inactivate restriction enzymes
   - added 0.1 microL (1 unit) of 10,000 units/mL CIP to vector DNA (R0010)
   - incubated vector DNA @ 37 dC 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

2. Image