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| ==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> |
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| ===Th 6.15.06===
| | *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.
| | ==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> |
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| ===W 6.14.06=== | | <calendar> |
| | name=IGEM:Harvard/2006/DNA_nanostructures/Notebook |
| | date=2006/10/01 |
| | view=threemonths |
| | format=%name/%year-%month-%day |
| | weekstart=7 |
| | </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 | | ==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=TChan/Notebook |
| | date=2006/2/01 |
| | view=threemonths |
| | format=%name/%year-%month-%day |
| | weekstart=7 |
| | </calendar> |
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| ====Digest Protocol==== | | <calendar> |
| | name=TChan/Notebook |
| | date=2006/5/01 |
| | view=threemonths |
| | format=%name/%year-%month-%day |
| | weekstart=7 |
| | </calendar> |
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| DIGEST PROTOCOL
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| ----------------
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| 8uL miniprepped DNA
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| 2.5uL 10x NEBuffer
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| 2.5uL 10x (diluted 1:10 from the 100x stock) BSA
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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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| 1uL enzyme2 (1:2)
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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°C 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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| ?
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| ==Contact Info== | | ==Contact Info== |
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| 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
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| [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] |
