IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-1

To do today

 * ordering
 * new oligo ligands
 * AscIII (if not already ordered
 * PEG precipitations
 * repeat experiment, run on PA gel, run on agarose gel
 * SYBR gold
 * read technical specs
 * determine lower threshold of imaging

Ordering
[[Media: v5latches_v3-2attacholigs_v5attacholigs_orderform_TC.xls|Order form for v5.0 latches, v3.2 attachment oligos, and v5.0 attachment oligos]]


 * v5.0 latches and v3.2 and v5.0 ligand-attachment oligos ordered
 * forgot to order the ligands - will do tomorrow, along with the clarified v4.0 ligand-attachment oligos

Trial 1

 * goal: to determine the minimum amount of DNA that can be visualized with SYBR Gold
 * methods: 12% PA gel with lanes listed below, run for 30 min. at 130 V, then stained with Invitrogen SYBR Gold.
 * prepared 1x solution: 10 thawed 10,000x SYBR gold in 100 mL TBE, stored in light-proof container at 4.


 * stained with 1x SYBR Gold for 50 min.
 * results
 * ladder stained brilliantly, visible both under SYBR Gold filter (and less so under EtBr filter)
 * no other DNAs showed, including after EtBr soak
 * dye was only halfway down gel, so ss oligos probably didn't run off
 * will try again with non biotinylated oligo

Trial 2

 * goal: try again, different DNA
 * run on August 2

Container 5.0 lid refolding

 * Yesterday's gel result indicated that folding two lids from a single scaffold didn't work well
 * Goal: fold c5.0 lids using separate scaffolds


 * Lid 1 seems to be causing the smearing
 * Lid 1+2 looks a lot better in this trial for some reason
 * Proper p7572 scaffold may improve things further

PEG precipitation

 * Goal: repeat titration of PEG precipitation conditions for folded containers
 * Katie and Val did one trial, Tiff and Matthew did another
 * Folding reactions
 * 2 samples of 6hb (100 final volume)
 * 4 samples of design 5 barrels (100 volume)
 * Mix the following
 * 10 uL 500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM
 * 40 uL working stock 250 nM each oligo (100 nM each oligo final concentration)
 * 50 uL p7308 20 nM (10 nM final concentration)
 * Anneal from 80 to 20, -1 per min


 * PEG precipitations
 * Plan to use 200 final volume
 * Trying 4%, 6%, 8%, 10%, 12%, 14%
 * Add 40 10 nM scaffold/100 nM oligo folded mix
 * Add 20% PEG solution (40, 60 , 80 , 100 , 120 , 140 )
 * Add 5 M NaCl stock solution (20 )
 * Add as much water to each as it takes to get them to a 200 final volume
 * Incubate on ice for 15 minutes
 * Spin 16k rcf for 10 minutes
 * Tiff and Matt's trial: 0.2 mL PCR tubes broke through the 1.5 mL microcentrifuge tubes that had been holding them. Will try again using 1.5 mL PCR tubes for reaction mixtures.'
 * Pipette out supernatant into separate tube
 * Resuspend pellet in 1x folding buffer volume equal to the supernatant
 * Run equivalent volumes of pellet and sup on 2% agarose gel (+ 11 mM MgCl2)


 * Gel Analysis
 * Analyze on 2% Mg agarose gel (0.5x TBE, 11 mM MgCl2, 0.5 μg/mL EtBr)
 * Load 1kb ladder (1 lane)
 * Load c5 supernatant, pellet for 0%, 4%, 6%, 8%, 10%, 12%, 14% (13 lanes)
 * Load 6hb supernatant, pellet for 0%, 4%, 14% (5 lanes)
 * 19 lanes total

Katie and Val's results:



High Concentrations of Nanoboxes, Day 2

 * Qiagen gel purified 3rxns of each Eb and Gb. Used 2% agarose Mg2+-supplemented gel.  Eluted each column (used 2/3rxns, so as to try to avoid overloading the column filter) into 40uL of EB, leaving 3rxns worth of nanoboxes (approx 200*3fmol, or 600fmol of biotin binding sites) in 80uL of solution.
 * scaffold (2nd lane) and folded Eb and Gb (3rd-14th lane) run differently - indicates folding worked