User:TChan

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==Week 1==
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<div class="tabs-blue">
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<ul>
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<li id="current">[[TChan|Project Overview]]</li>
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<li>[[TChan/Schematics|Schematics]]</li>
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<li>[[TChan/OligoList|Oligo List]]</li>
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<li>[[TChan/Scaffold|Scaffold]]</li>
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<li>[[TChan/Images|Images]]</li>
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<li>[[TChan/iGEMNotes|iGEM Notes]]</li>
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</ul>
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</div>
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<br style="clear:both">
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<br>
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===Th 6.15.06===
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*See [[IGEM:Harvard/2006/DNA_nanostructures|iGEM 2006 Page]] for more info
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1. RapidLigated 6uL of the insert (E0 fragments) and 1uL of the vector (R0 cut).
 
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2. Transformed and plated the entire 21uL of ligation reaction onto Carb plates.
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==iGEM 2006 Notebook==
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<calendar>
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name=IGEM:Harvard/2006/DNA_nanostructures/Notebook
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date=2006/07/01
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view=threemonths
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format=%name/%year-%month-%day
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weekstart=7
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</calendar>
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===W 6.14.06===
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<calendar>
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name=IGEM:Harvard/2006/DNA_nanostructures/Notebook
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date=2006/10/01
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view=threemonths
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format=%name/%year-%month-%day
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weekstart=7
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</calendar>
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<b>Goal:</b> Digest and extract out the E0-gfp and ligate after the R0-promoter, into that pre-cut plasmid.
 
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  R0: ---|E--|X---lacprom---|<b>S</b>--|<b>P</b>---
 
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  E0: ---|E--|<b>X</b>-----gfp-----|S--|<b>P</b>---
 
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  hypothetical ligation:
 
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  ------|E--|X---lacprom---|M-----gfp-----|P---
 
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1. Miniprepped the 3 BioBricks Plasmids (according to [http://openwetware.org/wiki/Miniprep/Qiagen_kit standard protocol])
 
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*Made glycerol stocks of 1mL of the cultures from each transformant:
 
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  666.6uL of 50% glycerol (to give 20% glycerol total)
 
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  1mL of transformant culture
 
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2. Double digested the 2 R0 (lac promoter) and 2 E0 (gfp) samples
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==Notebook==
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*NB: Due to the NanoSpec reading out negative DNA concentrations, we proceeded with an arbitrary amount of transformed DNA for the digests.
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<calendar>
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*Digested with SpeI and PstI (NEBuffer 2) for R0, XbaI and PstI (NEBuffer 3) for E0, with the following protocol:
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name=TChan/Notebook
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date=2006/2/01
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view=threemonths
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format=%name/%year-%month-%day
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weekstart=7
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</calendar>
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  DIGEST PROTOCOL
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<calendar>
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  ----------------
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name=TChan/Notebook
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  8uL miniprepped DNA
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date=2006/5/01
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  2.5uL 10x NEBuffer
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view=threemonths
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  2.5uL 10x (diluted 1:10 from the 100x stock) BSA
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format=%name/%year-%month-%day
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  1uL enzyme 1 (diluted 1:2 on Parafilm from the stock, of which we would have needed 0.5uL)
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weekstart=7
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  1uL enzyme2 (1:2)
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</calendar>
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  10uL dH2O
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  ----------------
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  For a TOTAL VOLUME of 25 uL
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  Incubated 1 hr @ 37%degC
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*Deactivated the enzymes by placing on 80%deg heat block for 15 min.
 
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3. Dephosphorylated 2 R0 samples to prevent self-ligation
 
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*Added 1 unit (1uL of 1:10 dilution from the 10,000U/mL stock, of which we would have needed 0.1uL) of CIP (Calf Intestine Phosphatase) to the 2 R0 reactions.
 
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*Incubated at 37%deg for 1hr.
 
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4. Ran R0 and E0 samples on gel to separate out the gfp from the E0-plasmid
 
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* Ran at 130V for ~45 min. 
 
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5. Gel extracted R0 plasmid (~2.1 kb, linearized by digestion) and E0 gfp fragment (~0.9kb)
 
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*Cut out the 1 bright E0 0.9kb band and the 2 bright R0 2.2kb bands
 
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*Extracted using the standard Qiagen protocol.
 
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*Froze overnight at -20%deg.
 
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===Tu 6.13.06===
 
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1. Picked Colonies of Existing BioBrick Transformants
 
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*Picked and numbered:
 
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  3 colonies of E7,
 
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  2 colonies of E0,
 
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  2 colonies of R0.
 
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*Grew each colony in 5mL LB + 5uL of 50mg/mL Amp (1:100 dilution).
 
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2. Ran Gel of DNA Nanostructures
 
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*Ran 2% agarose gel of the nanostructure reaction and the 2 controls for ~40 min at 130V.  (NB: Only left four lanes are ours.)
 
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  [[Image:Tc_mm_pt_lh_folding_0613.jpg|thumb|left|Nanostructure Gel]]
 
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  1kb Ladder | Oligo + Scaffold | -Oligo | -Scaffold
 
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===M 6.12.06===
 
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1. Folded DNA Nanostructures Using Shawn's Pre-designed Oligo Staples and Scaffold ssDNA
 
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*Three reactions below were mixed according to [http://openwetware.org/wiki/Folding_DNA_nanostructures  given protocol].
 
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oligos + scaffold
 
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-oligos
 
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-scaffold
 
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2. Transformed Bacteria with Existing BioBrick Plasmids
 
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*Transformed each BioBrick plasmid below into OneShot Top10 competent cells (Invitrogen) according to the following protocol.
 
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  R0010 (lac operon promoter),
 
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  E7104 (T4 promoter + gfp),
 
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  and E0241 (gfp).
 
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===Top10 Transformation===
 
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  Thaw cells; aliquot out ~30uL per transformation.
 
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  Add desired amount of plasmid DNA (1 uL in this case?) to cells and leave on ice for 30 min.
 
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  Heat shock at 37&degC for 30 sec in a heating block. 
 
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  Ice for 2 min.
 
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  Shake in 37%deg incubator for 1 hr.
 
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  Pour onto plates with the required antibiotic (carbinomycin in this case); spread with beads or plate spreader.
 
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  Incubate plates in 37%deg incubator overnight.
 
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HOMEWORK:
 
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# Past iGEM Projects Presentation: UCSF
 
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# Prospective Projects Research:
 
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  Lactobacillus Hijacking
 
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  Receptor-based DNA Nanostructure Latch
 
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  Biocryptography w/ DNA Nanostructures
 
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  ?
 
==Contact Info==
==Contact Info==
Line 113: Line 58:
   Biochemical Sciences
   Biochemical Sciences
   Kirkland '07
   Kirkland '07
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   PRISE Summer 2006
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   Academic Interests: DNA nanostructures, non-coding RNAs (SO. HOT.), drug design and delivery
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  Cell: 781 330 1969
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   [mailto:chan.tiffany@gmail.com chan.tiffany at gmail.com]
   [mailto:chan.tiffany@gmail.com chan.tiffany at gmail.com]
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  Currently taking [http://openwetware.org/wiki/Harvard:Biophysics_101/2007 Biophysics 101]

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Contact Info

 Tiffany Chan
 Biochemical Sciences
 Kirkland '07
 Academic Interests: DNA nanostructures, non-coding RNAs (SO. HOT.), drug design and delivery
 chan.tiffany at gmail.com
 Currently taking Biophysics 101
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