IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-15

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(Revised protection assay protocol (proposed))
(Revised protection assay protocol (proposed))
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* purify nanostructures with PEG precipitation using 7%, 8%, 9%, or 10%, depending on PEG fractionation repeat experiment
* purify nanostructures with PEG precipitation using 7%, 8%, 9%, or 10%, depending on PEG fractionation repeat experiment
-
''Digest''
+
'''Digest'''
* total volume of nanostructure digest: 20 {{ul}}
* total volume of nanostructure digest: 20 {{ul}}
** 100 fmols (~450 ng) purified, ligand-incubated nanostructures (MW ~= 4,500,000 Da) = 10 {{ul}} 10 nM
** 100 fmols (~450 ng) purified, ligand-incubated nanostructures (MW ~= 4,500,000 Da) = 10 {{ul}} 10 nM

Revision as of 15:28, 15 August 2006

Mg2+, Oligo-Concentration Titration w/ c5.0 (cont'd)

3. PEG

PEG:

  • GOAL: test 6%, 8%, 10% PEG precipitations
  • incubated on ice for 15 min.
  • spun at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
  • carefully pipetted off supernatant
  • resuspended "pellet" in 50 μL of water


Trial Final PEG % Lanes 20% PEG (μL) 5 M NaCl (μL) Nanostructures (μL) Water (μL) Total volume (μL)
1kb+ ladder-1----10
p7308-2----10
10%3 (untreated trial 1)-----
1-66%4 (pellet)155201050
1-66%5 (supernatant)-----
1-88%6 (pellet)20520550
1-88%7 (supernatant)-----
1-1010%8 (pellet)25520050
1-1010%9 (supernatant)-----
20%10 (untreated trial 2)-----
2-66%11 (pellet)155201050
2-66%12 (supernatant)-----
2-88%13 (pellet)20520550
2-88%14 (supernatant)-----
2-1010%15 (pellet)25520050
2-1010%16 (supernatant)-----
30%17 (untreated trial 3)-----
3-66%18 (pellet)155201050
3-66%19 (supernatant)-----
1kb+ ladder-1----10
p7308-2----10
3-88%3 (pellet)20520550
3-88%4 (supernatant)-----
3-1010%5 (pellet)25520050
3-1010%6 (supernatant)-----
40%7 (untreated trial 4)-----
4-66%8 (pellet)155201050
4-66%9 (supernatant)-----
4-88%10 (pellet)20520550
4-88%11 (supernatant)-----
4-1010%12 (pellet)25520050
4-1010%13 (supernatant)-----
50%14 (untreated trial 5)-----
5-66%15 (pellet)155201050
5-66%16 (supernatant)-----
5-88%17 (pellet)20520550
5-88%18 (supernatant)-----
5-1010%19 (pellet)25520050
5-1010%20 (supernatant)-----
1kb+ ladder-1----10
p7308-2----10
60%3 (untreated trial 6)----
6-66%4 (pellet)155201050
6-66%5 (supernatant)-----
6-88%6 (pellet)20520550
6-88%7 (supernatant)-----
6-1010%8 (pellet)25520050
6-1010%9 (supernatant)-----
6hb-44%10 (pellet)105201550
6hb-44%11 (supernatant)-----
6hb-66%12 (pellet)155201050
6hb-66%13 (supernatant)-----
6hb-88%14 (pellet)20520550
6hb-88%15 (supernatant)-----
6hb-1010%16 (pellet)25520050
6hb-1010%17 (supernatant)-----

Revised protection assay protocol (proposed)

Folding

  • use working stock that includes all latches, as well as oligo-ligand
  • fold nanostructures with appropriate folding conditions
    • appears to be either: 30 mM MgCl2 with 1x oligos or 20 mM MgCl2 with 6x, depending on PEG fractionation repeat experiment

Purification

  • purify nanostructures with PEG precipitation using 7%, 8%, 9%, or 10%, depending on PEG fractionation repeat experiment

Digest

  • total volume of nanostructure digest: 20 μL
    • 100 fmols (~450 ng) purified, ligand-incubated nanostructures (MW ~= 4,500,000 Da) = 10 μL 10 nM
    • 2 μL 10x BSA
    • 2 μL 10x NEBuffer 4
    • 5 μL water
    • 1 μL 500 units/mL AscI
  • negative control: 2.25 μL p7308 (or other molar amount of scaffold)
  • negative control: 10 μL 10 nM purified nanostructures
  • negative control: 1 μL 1 μM ligand DNA, 1 μL 1 μM attachment DNA, 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 14 μL water
  • positive control: 1 μL 1 μM ligand DNA, 1 μL 1 μM attachment DNA, 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 1 μL 500 units/mL AscI, 13 μL water
  • incubate all trials at 37[[:Category:{{{1}}}|{{{1}}}]] for 30 min.
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