IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-20

Design 5

 * Make working stock c5.0.A

Design 6

 * Mixing pre-working stocks (c6.0.1, c6.0.2, c6.0.3, c6.0.4, c6.0.5)
 * To mix pre-working stocks from plates, pipet 10 of each of the appropriate oligos into 1.5 mL tubes.
 * Mix working stocks c6.0.A, c6.0.B, c6.0.C

Pre-Working Stocks

Working Stock

Quantify p7308

 * Need to quantify p7308 to have enough scaffold to fold everything.
 * Pour 2% agraose, 11 mM gel
 * Run for 2 hrs, 70V
 * When imaging gel, use spot density tool to measure intensity of each band
 * Use saturation indicator to take a picture just below the point where any bands start saturating on the image
 * Draw a rectangle that fits around the largest band on the gel
 * Copy that rectangle and position it directly above the first band. This will be used to measure background
 * Repeat this for every band on the gel (one box for the band, one box for background)
 * Record this data along with gel picture on the wiki
 * To determine p7308 concentration, use background-subtracted value for each volume. Scale each unknown concentration against the control (44 nM) according to the ratio of background-subtracted intensity for the band that looks closest in intensity
 * For example, if the band in lane 1 (3, 44 nM) had an intensity of 1000, and the band in lane 5 (3 , ?? nM) had an intensity of 900, then we would record 900/1000 * 44 = 39.6 nM as the estimated concentration for that lane. Repeating for each lane should give you 3 data points, which you can average (throwing out any obvious outliers).

Folding protocol
16 ul oligos (from working stock) 9 ul p7308 11 ul H20 4 ul 10x folding buffer
 * Folding c5.0.A, c6.0.A, c6.0.B, c6.0.C - 24 tubes ea. * 40 per tube = 960  of each design
 * since new scaffold still needs to be quantified, will use older scaffold (44nM)
 * Each rxn:

PEG fractionation
40 20% PEG 10 5M NaCl 10 water 40 Nanostructures (add last) 50 20% PEG 10 5M NaCl 40 Nanostructures (add last)
 * Goal: to get cleaner purification of oligos away from nanostructures and to increase the volume of purified nanostructures we have for protection assays.
 * Nanostructures:
 * using 30mM, 1x oligos (seems to work best given previous results).
 * designs: c5.0.A, c5.0.C, c5.0.D key
 * PEG: 8%, 10%. Total volume in each is 100
 * 8 % Cocktail:
 * 10 % Cocktail:


 * incubate on ice for 15 min.
 * spin at 16 k rcf at 4 for 10 min.
 * carefully pipette off supernatant
 * resuspended "pellet" in 1x folding buffer. for now, resuspend in original total volume (100 but may resuspend in less in the future to improve protection assay results)
 * note: add PEG first, nanostructures last; mix using tapping after everything added. let sit for ~5 min. before putting it on ice