User:TChan

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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.
<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>---
+
*R0: ---|E--|X---lacprom---|<b>S</b>--|<b>P</b>---
-
  E0: ---|E--|<b>X</b>-----gfp-----|S--|<b>P</b>---
+
*E0: ---|E--|<b>X</b>-----gfp-----|S--|<b>P</b>---
    
    
-
  hypothetical ligation:  
+
Hypothetical Ligation:  
-
  ------|E--|X---lacprom---|M-----gfp-----|P---
+
*------|E--|X---lacprom---|M-----gfp-----|P---
1. Miniprepped the 3 BioBricks Plasmids (according to [http://openwetware.org/wiki/Miniprep/Qiagen_kit standard protocol])
1. Miniprepped the 3 BioBricks Plasmids (according to [http://openwetware.org/wiki/Miniprep/Qiagen_kit standard protocol])

Revision as of 18:01, 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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