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

Folding 5.0.A and 6.0

Design 5

• Make working stock c5.0.A

Working Stocks	Description	Pre-Working Stocks	Water	Total
c5.0A	no latches, no aptamers	1 (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 μL	200 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 oligos	box_v6_A; box_v6_B rows A-D (1-12), row E (1-9)	153
c6.0.2: barrel oligos @ outside aptamers -aptamers	box_v6_B row E (9-12), row F (1-12)	15
c6.0.3: barrel oligos @ inside aptamers -aptamers	box_v6_B row G (1-6)	6
c6.0.4: barrel oligos @ outside aptamers+ligand	box_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+ligand	box_v6_C row A (12), row B (1-12), row C (1-5)	18

Working Stock

Stock ID	Experiment	1	2	3	4	5
c6.0A	no aptamers	x	x	x	-	-
c6.0B	in aptamers	x	x	-	-	x
c6.0C	out aptamers	x	-	x	x	-

Working Stocks	Description	Pre-Working Stocks	Water	Total
c6.0A	no aptamers	1 (153 μL), 2 (15 μL), 3 (6 μL)	26 μL	200 μL
c6.0B	in aptamers	1 (153 μL), 2 (15 μL), 5 (18 μL)	14 μL	200 μL
c6.0C	out aptamers	1 (153 μL), 3 (6 μL), 4 (29 μL)	12 μL	200 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
0	1kb DNA ladder (5 μL)
1	p7308 060323, 44 nM (3 μL)	AGLB (2 μL) + dH2O (9 μL)
2	p7308 060323, 44 nM (6 μL)	AGLB (2 μL) + dH2O (6 μL)
3	p7308 060323, 44 nM (9 μL)	AGLB (2 μL) + dH2O (3 μL)
4	p7308 060522, (1 μL)	AGLB (2 μL) + dH2O (11 μL)
5	p7308 060522, (3 μL)	AGLB (2 μL) + dH2O (9 μL)
6	p7308 060522, 6 μL)	AGLB (2 μL) + dH2O (6 μL)
7	p7308 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