User:TChan

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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.
*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:
*Digested with SpeI and PstI (NEBuffer 2) for R0, XbaI and PstI (NEBuffer 3) for E0, with the following protocol:
 +
 +
====Digest Protocol====
   DIGEST PROTOCOL
   DIGEST PROTOCOL
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   and E0241 (gfp).
   and E0241 (gfp).
-
===Top10 Transformation===
+
====Top10 Transformation====
   Thaw cells; aliquot out ~30uL per 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.
   Add desired amount of plasmid DNA (1 uL in this case?) to cells and leave on ice for 30 min.

Revision as of 18:00, 15 June 2006

Contents

Week 1

Th 6.15.06

1. RapidLigated 6uL of the insert (E0 fragments) and 1uL of the vector (R0 cut).

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

W 6.14.06

Goal: Digest and extract out the E0-gfp and ligate after the R0-promoter, into that pre-cut plasmid.

 R0: ---|E--|X---lacprom---|S--|P---
 E0: ---|E--|X-----gfp-----|S--|P---
 
 hypothetical ligation: 
 ------|E--|X---lacprom---|M-----gfp-----|P---

1. Miniprepped the 3 BioBricks Plasmids (according to 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.)
Nanostructure Gel
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

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:

  1. Past iGEM Projects Presentation: UCSF
  2. Prospective Projects Research:
 Lactobacillus Hijacking
 Receptor-based DNA Nanostructure Latch 
 Biocryptography w/ DNA Nanostructures
 ?

Contact Info

 Tiffany Chan
 Biochemical Sciences
 Kirkland '07
 PRISE Summer 2006
 Cell: 781 330 1969
 chan.tiffany at gmail.com
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