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:
- For a total volume of 25 uL:
3. Phosphatasing of 2 R0 samples to prevent self-ligation
4. Ran R0 and E0 samples on gel to separate out the gfp from the E0-plasmid
5. Gel extracted R0 plasmid (~2.1 kb, linearized by digestion) and E0 gfp fragment (~0.9kb)
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.)
1kb Ladder | Oligo + Scaffold | -Oligo | -Scaffold
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).
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°C 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.
- Past iGEM Projects Presentation: UCSF
- Prospective Projects Research:
Lactobacillus Hijacking Receptor-based DNA Nanostructure Latch Biocryptography w/ DNA Nanostructures ?
Tiffany Chan Biochemical Sciences Kirkland '07 PRISE Summer 2006 Cell: 781 330 1969 chan.tiffany at gmail.com