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

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(Quantitation)
(3. PEG)
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* carefully pipetted off supernatant
* carefully pipetted off supernatant
* resuspended "pellet" in 50 {{ul}} of water
* resuspended "pellet" in 50 {{ul}} of water
 +
**note: we added PEG first, nanostructures last; mixed using tapping after everything was added; let it sit for like...5 min. before putting it on ice; let it sit a bit (ie. 10 min.) after centrifuging
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Revision as of 16:29, 15 August 2006

Contents

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
    • note: we added PEG first, nanostructures last; mixed using tapping after everything was added; let it sit for like...5 min. before putting it on ice; let it sit a bit (ie. 10 min.) after centrifuging

RESULTS:

  • decent separation of pellet and oligos for most of the trials
  • looking at gels after 75 min. seems to show that the nanostructures are going into the gel, so we don't think they're getting denatured
  • best trials seem to be: Trial 4 (20 mM MgCl2 w/ 6x oligos) and Trial 5 (30 mM MgCl2 w/ 1x oligos), each at 8% and 10% PEG
  • next step: we will repeat this experiment in larger quantities to prepare for a protection assay


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 each digest trial: 20 μL
trial DNA 10x NEBuffer 4 10x BSA AscI water
experimental10 μL 10 nM purified, ligand-incubated nanostructures2 μL2 μL1 μL 500 U/mL5 μL
-oligos2.25 μL 44 nM p7308 (or appropriate scaffold)2 μL2 μL1 μL 500 U/mL13.75 μL
-enzyme10 μL 10 nM purified, ligand-incubated nanostructures2 μL2 μL0 μL6 μL
-ligand10 μL 10 nM purified nanostructures (no ligand)2 μL2 μL1 μL 500 U/mL5 μL
-nanostructures -enzyme1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand2 μL2 μL0 μL14 μL
-nanostructures1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand2 μL2 μL1 μL 500 U/mL13 μL

Quantitation

p7308 quantitation

  • Speedvac 060522 p7308 sample down to 50% volume. This should remove any ethanol, and give you a slightly more manageable volume.
  • Pour 2% agraose, 11 mM MgCl2 gel
  • For gel loading make 1:2 dilution (add 20 μL of p7308 to 20 μL dH2O). Original estimate for 060522 prep was 42 nM, so hopefully it should correspond pretty well to 44 nM sample.
  • Load gel according to table below
  • 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).
2% agarose gel, 0.5 mg/mL EtBr0.5x TBE, 11 mM MgCl2
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
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, 1:2 dil (1 μL)AGLB (2 μL) + dH2O (11 μL)
5p7308 060522, 1:2 dil (3 μL)AGLB (2 μL) + dH2O (9 μL)
6p7308 060522, 1:2 dil (6 μL)AGLB (2 μL) + dH2O (6 μL)
7p7308 060522, 1:2 dil (9 μL)AGLB (2 μL) + dH2O (3 μL)
8p7308 060522, 1:2 dil (12 μL)AGLB (2 μL)
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