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| ==Contact Info.== | | <div class="tabs-blue"> |
| vlau@fas.harvard.edu/vhtlau@gmail.com | | <ul> |
| | <li id="current">[[IGEM:Harvard/2006/vlau|Contact Info.]]</li> |
| | <li>[[IGEM:Harvard/2006/vlau/Lab Notebook|Lab Notebook]]</li> |
| | <li>[[IGEM:Harvard/2006/vlau/Presentations|Presentations]]</li> |
| | <li>[[IGEM:Harvard/2006/vlau/Protocols|Protocols]]</li> |
| | </ul> |
| | </div> |
| | <br style="clear:both"> |
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| [[iGEM:Harvard/2006]]
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| ==[[Week 1]]==
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| ==='''06.12.06: Mon.'''===
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| ====''Folding DNA Nanostructure''s====
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| 1. Working Stocks
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| 44 nM scaffold (20 microL)
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| 0.99 microM of each oligo
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| 2. Protocol
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| - goal: 10 nM scaffold w/ 100 nM each oligo, total rxn vol of 20 microL
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| - calculations:
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| scaffold = (10 nM)/(44 nM) * 20 microL = 4.5 microL
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| oligos = (100 nM)/(990 nM) * 20 microL = 2 microL
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| - reaction mixture:
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| 4.5 microL p7308 scaffold
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| 2 microL oligos
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| 2 microL 10x folding buffer (500 mM HEPES ph 7.5, 500 mM NaCl, 100 mM MgCl2)
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| 11.5 microL dH2O
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| - annealing times:
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| 90 dC, 5'
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| 65 dC, 20'
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| 55 dC, 20'
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| 45 dC, 20'
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| 37 dC, 30'
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| - neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water
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| ====''Transformation''====
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| 1. Plasmids
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| R0010: lac operon promoter
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| E7104: T7 promoter + GFP
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| E0241: GFP
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| 2. Protocol
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| - let chemically competent OneShot Top10 cells thaw on ice
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| - added appropriate DNA to cells and tapped gently to mix
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| - let sit on ice for 20 min
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| - heat shocked @ 42 dC for 30"
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| - let cool on ice for 2 min
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| - added 200 microL SOC media
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| - shook @ 37 dC for 1 hr
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| - pipetted and spread onto agar plates treated w/ ?
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| - incubated @ 37 dC overnight agar side-up
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| ==='''06.13.06: Tues.'''===
| | 335 Kirkland House Mail Center |
| ====''Gel Electrophoresis w/ 2% Gel''====
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| 1. Protocol
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| - mixed 2 g Ultrapure agarose w/ 100 mL 1X TBE in plastic flask
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| - heated for 1.5 min in microwave w/ top loosely screwed on
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| - added 3 microL EtBr
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| - poured into gel frame and let set for 20 min
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| - placed into running box and submerged w/ TBE
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| - wells loaded
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| - ran gel @ 130 V, 45 min
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| 2. Samples
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| L. 1: 1 kB ladder
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| L. 2 and 3: DNA nanostructure
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| L. 4 and 5: neg control w/ no oligonucleotides
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| L. 6 and 7: neg control w/ no scaffold
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| L. 8: 1 kB ladder
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| loading dye: BTB dye 50% glycerol + 10X TBE
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| ladder: 10 microL + 1 microL dye
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| samples: 10 microL + 1 microL dye
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| 3. Image
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| [[Image:Pic 1.jpg|thumb|center]]
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| ====''Transformation P. 2''====
| | vlau@fas.harvard.edu | vhtlau@gmail.com |
| 1. Protocol
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| - prepared 2 liquid cultures for each transformation
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| - added 50 microL of 5 mg/mL amp to 5 mL LB per culture
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| - shook @ 180 rpm, 37 dC overnight
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| ==='''06.14.06: Wed.'''===
| | [[IGEM:Harvard/2006]] |
| ====''Miniprep''====
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| 1. Plasmids (2 samples each)
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| R0010: lac operon promoter
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| E7104: T7 promoter + GFP
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| E0241: GFP
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| 2. Protocol
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| - w/ 5 mL samples, 1 mL saved for glycerol stock
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| - rest of samples transferred to 1.5 eppendorfs and centrifuged to form pellet
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| - supernatant removed and pellet resuspended in 250 microL Buffer P1
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| - 250 microL Buffer P2 added and tube inverted 4-6 times for mixing
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| (no vortexing to prevent shearing of genomic DNA; let sit for no longer than 5 min)
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| - 350 microL Buffer N3 added and tube inverted 4-6 times for mixing
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| - centrifuged @ 13,000 rpm for 10 min
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| (formation of white pellet)
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| - supernatant transferred to QIAprep spin column and centrifuged for 1 min
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| - 0.5 mL Buffer PB added and centrifuged for 1 min
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| (optional)
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| - added 0.75 mL Buffer PE for washing and centrifuged for 1 min
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| - flowthrough discarded and centrifuged for 1 min to remove residual buffer
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| - QIAprep column transferred to 1.5 eppendorfs
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| - 50 microL water added to center of column
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| (let column stand for 1 min)
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| - centrifuged for 1 min
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| - nanodropped
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| ====''Digestion''====
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| 1. Materials
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| 8 microL DNA (R0010, E0241)
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| 2.5 microL 10x BSA
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| 2.5 microL 10x Buffer (Buffer 2)
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| 11 microL dH2O
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| 0.5 microL enzyme 1 and 2 (SpeI and PstI, XbaI and PstI)
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| total vol = 25 microL
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| 2. Protocol
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| - checked www.neb.com to determine correct Buffer
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| - digested @ 37 dC for 1 hr
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| - heat shocked @ 97 dc for 15 min to inactivate restriction enzymes
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| - added 0.1 microL (1 unit) of 10,000 units/mL CIP to vector DNA (R0010)
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| - incubated vector DNA @ 37 dC for 1 hr
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| ====''Gel Electrophoresis and Purification''====
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| 1. 1.0% Agarose Gel
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| L. 1: 1 kb Ladder
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| L. 2: R0010 Sample 1
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| L. 3: R0010 Sample 2
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| L. 4: E0241 Sample 1
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| L. 5: E0241 Sample 2
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| 2. Image
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| [[Image:Pic 2.jpg|thumb|center]]
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| ==='''06.15.06: Thurs.'''===
| | [[IGEM:Harvard/2006/DNA nanostructures]] |
| ====''Ligation''====
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| ====''Transformation''====
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| ==Week 2==
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| ==='''06.19.06: Mon.'''===
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| ====''Presentation: Project Proposal''====
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| [[Media:Week 2 Presentation.ogg]] | |
