User:TChan: Difference between revisions

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==W 6.14.06==
<div class="tabs-blue">
<ul>
<li id="current">[[TChan|Project Overview]]</li>
<li>[[TChan/Schematics|Schematics]]</li>
<li>[[TChan/OligoList|Oligo List]]</li>
<li>[[TChan/Scaffold|Scaffold]]</li>
<li>[[TChan/Images|Images]]</li>
<li>[[TChan/iGEMNotes|iGEM Notes]]</li>
</ul>
</div>
<br style="clear:both">
<br>


<b>Goal:</b> Digest and extract out the E0-gfp and ligate after the R0-promoter, into that pre-cut plasmid.
*See [[IGEM:Harvard/2006/DNA_nanostructures|iGEM 2006 Page]] for more info
 
  R0: ---|E--|X---lacprom---|<b>S</b>--|<b>P</b>---
  E0: ---|E--|<b>X</b>-----gfp-----|S--|<b>P</b>---
  ---
  hypothetical ligation:
  ------|E--|X---lacprom---|M-----gfp-----|P---


1. Miniprepped the 3 BioBricks Plasmids (according to [http://openwetware.org/wiki/Miniprep/Qiagen_kit standard protocol])
*Made glycerol stocks of 1mL of the cultures from each transformant:
  666.6uL of 50% glycerol (to give 20% glycerol total)
  1mL of transformant culture


2. Double digested the 2 R0 (lac promoter) and 2 E0 (gfp) samples
==iGEM 2006 Notebook==
*NB: Due to the NanoSpec reading out negative DNA concentrations, we proceeded with an arbitrary amount of transformed DNA for the digests.
<calendar>
*Digested with SpeI and PstI (NEBuffer 2) for R0, XbaI and PstI (NEBuffer 3) for E0, with the following protocol:
name=IGEM:Harvard/2006/DNA_nanostructures/Notebook
date=2006/07/01
view=threemonths
format=%name/%year-%month-%day
weekstart=7
</calendar>


  DIGEST PROTOCOL
<calendar>
  ----------------
name=IGEM:Harvard/2006/DNA_nanostructures/Notebook
  8uL miniprepped DNA
date=2006/10/01
  2.5uL 10x NEBuffer
view=threemonths
  2.5uL 10x (diluted 1:10 from the 100x stock) BSA
format=%name/%year-%month-%day
  1uL enzyme 1 (diluted 1:2 on Parafilm from the stock, of which we would have needed 0.5uL)
weekstart=7
  1uL enzyme2 (1:2)
</calendar>
  10uL dH2O
  ----------------
  For a TOTAL VOLUME of 25 uL
  Incubated 1 hr @ 37%degC


*Deactivated the enzymes by placing on 80%deg heat block for 15 min.


3. Phosphatasing of 2 R0 samples to prevent self-ligation
*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.
*Incubated at 37%deg for 1hr.


4. Ran R0 and E0 samples on gel to separate out the gfp from the E0-plasmid
==Notebook==
* Ran at 130V for ~45 min. 
<calendar>
name=TChan/Notebook
date=2006/2/01
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format=%name/%year-%month-%day
weekstart=7
</calendar>


5. Gel extracted R0 plasmid (~2.1 kb, linearized by digestion) and E0 gfp fragment (~0.9kb)
<calendar>
*Cut out the 1 bright E0 0.9kb band and the 2 bright R0 2.2kb bands
name=TChan/Notebook
*Extracted using the standard Qiagen protocol.
date=2006/5/01
view=threemonths
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weekstart=7
</calendar>


*Froze overnight at -20%deg.


==Tu 6.13.06==
1. Picked Colonies of Existing BioBrick Transformants
*Picked and numbered:
  3 colonies of E7,
  2 colonies of E0,
  2 colonies of R0.
*Grew each colony in 5mL LB + 5uL of 50mg/mL Amp (1:100 dilution).


2. Ran Gel of DNA Nanostructures
*Ran 2% agarose gel of the nanostructure reaction and the 2 controls for ~40 min at 130V.  (NB: Only left four lanes are ours.)
  [[Image:Tc_mm_pt_lh_folding_0613.jpg|thumb|left|Nanostructure Gel]]
 
  1kb Ladder | Oligo + Scaffold | -Oligo | -Scaffold
==M 6.12.06==
1. Folded DNA Nanostructures Using Shawn's Pre-designed Oligo Staples and Scaffold ssDNA
*Three reactions below were mixed according to [http://openwetware.org/wiki/Folding_DNA_nanostructures  given protocol].
oligos + scaffold
-oligos
-scaffold
2. Transformed Bacteria with Existing BioBrick Plasmids
*Transformed each BioBrick plasmid below into OneShot Top10 competent cells (Invitrogen) according to the following protocol.
  R0010 (lac operon promoter),
  E7104 (T4 promoter + gfp),
  and E0241 (gfp).
===Top10 Transformation===
  Thaw cells; aliquot out ~30uL per transformation.
  Add desired amount of plasmid DNA (1 uL in this case?) to cells and leave on ice for 30 min.
  Heat shock at 37&degC for 30 sec in a heating block. 
  Ice for 2 min.
  Shake in 37%deg incubator for 1 hr.
  Pour onto plates with the required antibiotic (carbinomycin in this case); spread with beads or plate spreader.
  Incubate plates in 37%deg incubator overnight.
HOMEWORK:
# Past iGEM Projects Presentation: UCSF
# Prospective Projects Research:
  Lactobacillus Hijacking
  Receptor-based DNA Nanostructure Latch
  Biocryptography w/ DNA Nanostructures
  ?


==Contact Info==
==Contact Info==
Line 105: Line 58:
   Biochemical Sciences
   Biochemical Sciences
   Kirkland '07
   Kirkland '07
   PRISE Summer 2006
   Academic Interests: DNA nanostructures, non-coding RNAs (SO. HOT.), drug design and delivery
  Cell: 781 330 1969
   [mailto:chan.tiffany@gmail.com chan.tiffany at gmail.com]
   [mailto:chan.tiffany@gmail.com chan.tiffany at gmail.com]
  Currently taking [http://openwetware.org/wiki/Harvard:Biophysics_101/2007 Biophysics 101]

Latest revision as of 13:12, 9 April 2009




iGEM 2006 Notebook

<calendar> name=IGEM:Harvard/2006/DNA_nanostructures/Notebook date=2006/07/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>

<calendar> name=IGEM:Harvard/2006/DNA_nanostructures/Notebook date=2006/10/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>


Notebook

<calendar> name=TChan/Notebook date=2006/2/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>

<calendar> name=TChan/Notebook date=2006/5/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>



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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