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.
  • List of pre-working and working stock oligos: Table_of_c6.0_Working_Stocks
• Mix working stocks c6.0.A, c6.0.B, c6.0.C

PEG precipitation

• 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 50 μL of water
  • note: add PEG first, nanostructures last; mix using tapping after everything added. let sit for ~5 min. before putting it on ice