Difference between revisions of "IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-15"

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(Revised protection assay protocol (proposed))
(Revised protection assay protocol (proposed))
Line 159: Line 159:
 
** 5 {{ul}} water
 
** 5 {{ul}} water
 
** 1 {{ul}} 500 units/mL AscI
 
** 1 {{ul}} 500 units/mL AscI
* negative control: 2.25 {{ul}} p7308 (or other molar amount of scaffold)
+
* negative control: 2.25 {{ul}} p7308 (or other molar amount of scaffold), 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 13.75 {{ul}} water
* negative control: 10 {{ul}} 10 nM purified nanostructures
+
* negative control: 10 {{ul}} 10 nM purified nanostructures, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 6 {{ul}} water
 +
* negative control: 10 {{ul}} 10 nM purified nanostructures (with no oligo-ligand added), 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 1 {{ul}} 500 units/mL AscI, 5 {{ul}} water
 
* negative control: 1 {{ul}} 1 {{um}} ligand DNA, 1 {{ul}} 1 {{um}} attachment DNA, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 14 {{ul}} water
 
* negative control: 1 {{ul}} 1 {{um}} ligand DNA, 1 {{ul}} 1 {{um}} attachment DNA, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 14 {{ul}} water
 
* positive control: 1 {{ul}} 1 {{um}} ligand DNA, 1 {{ul}} 1 {{um}} attachment DNA, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 1 {{ul}} 500 units/mL AscI, 13 {{ul}} water
 
* positive control: 1 {{ul}} 1 {{um}} ligand DNA, 1 {{ul}} 1 {{um}} attachment DNA, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 1 {{ul}} 500 units/mL AscI, 13 {{ul}} water
 
* incubate all trials at 37{{c}} for 30 min.
 
* incubate all trials at 37{{c}} for 30 min.

Revision as of 12:32, 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
1 0% 3 (untreated trial 1) - - - - -
1-6 6% 4 (pellet) 15 5 20 10 50
1-6 6% 5 (supernatant) - - - - -
1-8 8% 6 (pellet) 20 5 20 5 50
1-8 8% 7 (supernatant) - - - - -
1-10 10% 8 (pellet) 25 5 20 0 50
1-10 10% 9 (supernatant) - - - - -
2 0% 10 (untreated trial 2) - - - - -
2-6 6% 11 (pellet) 15 5 20 10 50
2-6 6% 12 (supernatant) - - - - -
2-8 8% 13 (pellet) 20 5 20 5 50
2-8 8% 14 (supernatant) - - - - -
2-10 10% 15 (pellet) 25 5 20 0 50
2-10 10% 16 (supernatant) - - - - -
3 0% 17 (untreated trial 3) - - - - -
3-6 6% 18 (pellet) 15 5 20 10 50
3-6 6% 19 (supernatant) - - - - -
1kb+ ladder - 1 - - - - 10
p7308 - 2 - - - - 10
3-8 8% 3 (pellet) 20 5 20 5 50
3-8 8% 4 (supernatant) - - - - -
3-10 10% 5 (pellet) 25 5 20 0 50
3-10 10% 6 (supernatant) - - - - -
4 0% 7 (untreated trial 4) - - - - -
4-6 6% 8 (pellet) 15 5 20 10 50
4-6 6% 9 (supernatant) - - - - -
4-8 8% 10 (pellet) 20 5 20 5 50
4-8 8% 11 (supernatant) - - - - -
4-10 10% 12 (pellet) 25 5 20 0 50
4-10 10% 13 (supernatant) - - - - -
5 0% 14 (untreated trial 5) - - - - -
5-6 6% 15 (pellet) 15 5 20 10 50
5-6 6% 16 (supernatant) - - - - -
5-8 8% 17 (pellet) 20 5 20 5 50
5-8 8% 18 (supernatant) - - - - -
5-10 10% 19 (pellet) 25 5 20 0 50
5-10 10% 20 (supernatant) - - - - -
1kb+ ladder - 1 - - - - 10
p7308 - 2 - - - - 10
6 0% 3 (untreated trial 6) - - - -
6-6 6% 4 (pellet) 15 5 20 10 50
6-6 6% 5 (supernatant) - - - - -
6-8 8% 6 (pellet) 20 5 20 5 50
6-8 8% 7 (supernatant) - - - - -
6-10 10% 8 (pellet) 25 5 20 0 50
6-10 10% 9 (supernatant) - - - - -
6hb-4 4% 10 (pellet) 10 5 20 15 50
6hb-4 4% 11 (supernatant) - - - - -
6hb-6 6% 12 (pellet) 15 5 20 10 50
6hb-6 6% 13 (supernatant) - - - - -
6hb-8 8% 14 (pellet) 20 5 20 5 50
6hb-8 8% 15 (supernatant) - - - - -
6hb-10 10% 16 (pellet) 25 5 20 0 50
6hb-10 10% 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), 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 13.75 μL water
  • negative control: 10 μL 10 nM purified nanostructures, 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 6 μL water
  • negative control: 10 μL 10 nM purified nanostructures (with no oligo-ligand added), 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 1 μL 500 units/mL AscI, 5 μL water
  • 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.