User:TChan: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
 
(27 intermediate revisions by the same user not shown)
Line 1: Line 1:
==Week 1==
<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>


===F 6.16.06===
*See [[IGEM:Harvard/2006/DNA_nanostructures|iGEM 2006 Page]] for more info
====Relevant Papers Summaries====


<biblio>
rps1 pmid=8407909</biblio><br>


Thrombin, a serine protease functioning in the blood clotting pathways, is bound by a DNA aptamer with the sequence GGTTGGTGTGGTTGG; this binding inactivates thrombin.  Assays of the aptamer's binding site and specificity were done, showing that the aptamer binds thrombin over competitive analogues.  Assays also showed that the aptamer binds thrombin subunit alpha-thrombin rather than gamma-thrombin, which is the active-site-containing part of thrombin - in other words, the aptamer doesn't change the functionality of the active site, but it does change the ability of the protein to bind its substrate in the necessary way for cleavage.  The group pinpointed the binding activity to two specific lysine residues in the protein, Lys 21 and 65. 
==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>


<biblio>
rps2 pmid=16493417</biblio><br>


MscL and MscS are mechanosensitive ion channels, named for their respective large or small conductances.  Their mechanosensitivity to lateral distension across the lipid bilayer They are in effect a 'last ditch response' to physical stress which might result in fatal osmolarity changes.  MscL is a five subunit protein, each subunit being comprised of two transmembrane helices; MscS is also a five subunit protein, each subunit comprised of transmembrane helices with a one cytosolic section.  MscL has a 2-angstrom diameter pore when closed, but MscS has been resolved with an 11-angstrom pore - whether this is true when MscS is in open or closed state has still to be determined, due to water dynamics studies showing that in this 11-angstrom wide state, the hydrophobic residues may from a "vapor lock," preventing any solubilized ion from passing through.


===Th 6.15.06===
==Notebook==
<calendar>
name=TChan/Notebook
date=2006/2/01
view=threemonths
format=%name/%year-%month-%day
weekstart=7
</calendar>


1. RapidLigated 6uL of the insert (E0 fragments) and 1uL of the vector (R0 cut).
<calendar>
name=TChan/Notebook
date=2006/5/01
view=threemonths
format=%name/%year-%month-%day
weekstart=7
</calendar>


2. Transformed and plated the entire 21uL of ligation reaction onto Carb plates.


===W 6.14.06===


<b>Goal:</b> Digest and extract out the E0-gfp and ligate after the R0-promoter, into that pre-cut plasmid.
 
*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
*NB: Due to the NanoSpec reading out negative DNA concentrations, we proceeded with an arbitrary amount of transformed DNA for the digests.
*Digested with SpeI and PstI (NEBuffer 2) for R0, XbaI and PstI (NEBuffer 3) for E0, with the following protocol:
====Digest Protocol====
  DIGEST PROTOCOL
  ----------------
  8uL miniprepped DNA
  2.5uL 10x NEBuffer
  2.5uL 10x (diluted 1:10 from the 100x stock) BSA
  1uL enzyme 1 (diluted 1:2 on Parafilm from the stock, of which we would have needed 0.5uL)
  1uL enzyme2 (1:2)
  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. Dephosphorylated 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
* Ran at 130V for ~45 min. 
5. Gel extracted R0 plasmid (~2.1 kb, linearized by digestion) and E0 gfp fragment (~0.9kb)
*Cut out the 1 bright E0 0.9kb band and the 2 bright R0 2.2kb bands
*Extracted using the standard Qiagen protocol.
*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 129: Line 58:
   Biochemical Sciences
   Biochemical Sciences
   Kirkland '07
   Kirkland '07
   PRISE Summer 2006, Room D-23
   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
Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:TChan&oldid=300657"