User:TChan

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
 ----------------
 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

 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 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:

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