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

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Contents

Folding 5.0.A and 6.0

Design 5

  • Make working stock c5.0.A
Working Stocks Description Pre-Working Stocks Water Total
c5.0Ano latches, no aptamers1 (86 μL), 2 (49 μL), 3 (49 μL), 4 (2.5 μL), 5 (2.5 μL), 6 (1.5 μL), 7 (1.5 μL)8 μL200 μL

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 μL 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

Description Plate Locations Total
c6.0.1: core barrel oligosbox_v6_A; box_v6_B rows A-D (1-12), row E (1-9)153
c6.0.2: barrel oligos @ outside aptamers -aptamersbox_v6_B row E (9-12), row F (1-12)15
c6.0.3: barrel oligos @ inside aptamers -aptamersbox_v6_B row G (1-6)6
c6.0.4: barrel oligos @ outside aptamers+ligandbox_v6_B row G (7-12), row H (1-12); box_v6_C row A (1-11)29
c6.0.5: barrel oligos @ inside aptamers+ligandbox_v6_C row A (12), row B (1-12), row C (1-5)18

Working Stock

Stock IDExperiment12345
c6.0Ano aptamersxxx--
c6.0Bin aptamersxx--x
c6.0Cout aptamersx-xx-
Working Stocks Description Pre-Working Stocks Water Total
c6.0Ano aptamers1 (153 μL), 2 (15 μL), 3 (6 μL)26 μL200 μL
c6.0Bin aptamers1 (153 μL), 2 (15 μL), 5 (18 μL)14 μL200 μL
c6.0Cout aptamers1 (153 μL), 3 (6 μL), 4 (29 μL)12 μL200 μL

Quantify p7308

  • Need to quantify p7308 to have enough scaffold to fold everything.
  • Pour 2% agraose, 11 mM MgCl2 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 μL, 44 nM) had an intensity of 1000, and the band in lane 5 (3 μL, ?? 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).
Lane Contents Loading Buffer
01kb DNA ladder (5 μL)
1p7308 060323, 44 nM (3 μL)AGLB (2 μL) + dH2O (9 μL)
2p7308 060323, 44 nM (6 μL)AGLB (2 μL) + dH2O (6 μL)
3p7308 060323, 44 nM (9 μL)AGLB (2 μL) + dH2O (3 μL)
4p7308 060522, (1 μL)AGLB (2 μL) + dH2O (11 μL)
5p7308 060522, (3 μL)AGLB (2 μL) + dH2O (9 μL)
6p7308 060522, 6 μL)AGLB (2 μL) + dH2O (6 μL)
7p7308 060522, (9 μL)AGLB (2 μL) + dH2O (3 μL)


Folding protocol

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

PEG fractionation

  • 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 MgCl2, 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 μL
    • 8 % Cocktail:
     40 μL 20% PEG
     10 μL 5M NaCl
     10 μL water
     40 μL Nanostructures (add last)
    • 10 % Cocktail:
     50 μL 20% PEG
     10 μL 5M NaCl
     40 μL Nanostructures (add last)
  • incubate on ice for 15 min.
  • spin at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
  • carefully pipette off supernatant
  • resuspended "pellet" in 1x folding buffer. for now, resuspend in original total volume (100 μL 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
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