User:Vlau/Lab Notebook/Week 1

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

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

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