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

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m (Quantitation)
(Quantitation)
Line 182: Line 182:
  
 
==Quantitation==
 
==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 {{ul}} of p7308 to 20 {{ul}} d{{h2o}}). 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 {{ul}}, 44 nM) had an intensity of 1000, and the band in lane 5 (3 {{ul}}, ?? 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).
  
continued from [[http://openwetware.org/wiki/IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-11#p7308_quantitation|Aug. 12]]
+
[[Image:Igemharv06_20060815_quant.tif|thumb|2% agarose gel, 0.5 mg/mL EtBr<br>0.5x TBE, 11 mM {{mgcl2}}]]
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Lane'''
 +
| align="center" style="background:#f0f0f0;"|'''Contents'''
 +
| align="center" style="background:#f0f0f0;"|'''Loading Buffer'''
 +
|-
 +
| 0||1kb DNA ladder (5 {{ul}})||
 +
|-
 +
| 1||p7308 060323, 44 nM (3 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (9 {{ul}})
 +
|-
 +
| 2||p7308 060323, 44 nM (6 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (6 {{ul}})
 +
|-
 +
| 3||p7308 060323, 44 nM (9 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (3 {{ul}})
 +
|-
 +
| 4||p7308 060522, 1:2 dil (1 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (11 {{ul}})
 +
|-
 +
| 5||p7308 060522, 1:2 dil (3 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (9 {{ul}})
 +
|-
 +
| 6||p7308 060522, 1:2 dil (6 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (6 {{ul}})
 +
|-
 +
| 7||p7308 060522, 1:2 dil (9 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (3 {{ul}})
 +
|-
 +
| 8||p7308 060522, 1:2 dil (12 {{ul}})||AGLB (2 {{ul}})
 +
|}

Revision as of 12:23, 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

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
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 each digest trial: 20 μL
trial DNA 10x NEBuffer 4 10x BSA AscI water
experimental 10 μL 10 nM purified, ligand-incubated nanostructures 2 μL 2 μL 1 μL 500 U/mL 5 μL
-oligos 2.25 μL 44 nM p7308 (or appropriate scaffold) 2 μL 2 μL 1 μL 500 U/mL 13.75 μL
-enzyme 10 μL 10 nM purified, ligand-incubated nanostructures 2 μL 2 μL 0 μL 6 μL
-ligand 10 μL 10 nM purified nanostructures (no ligand) 2 μL 2 μL 1 μL 500 U/mL 5 μL
-nanostructures -enzyme 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand 2 μL 2 μL 0 μL 14 μL
-nanostructures 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand 2 μL 2 μL 1 μL 500 U/mL 13 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).

File:Igemharv06 20060815 quant.tif

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, 1:2 dil (1 μL) AGLB (2 μL) + dH2O (11 μL)
5 p7308 060522, 1:2 dil (3 μL) AGLB (2 μL) + dH2O (9 μL)
6 p7308 060522, 1:2 dil (6 μL) AGLB (2 μL) + dH2O (6 μL)
7 p7308 060522, 1:2 dil (9 μL) AGLB (2 μL) + dH2O (3 μL)
8 p7308 060522, 1:2 dil (12 μL) AGLB (2 μL)