Difference between revisions of "IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-11"

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(Gigundo PEG precipitation)
(Gigundo PEG precipitation)
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Protocol: prepare the following 30 samples.
 
Protocol: prepare the following 30 samples.
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[[Image:2006-08-11_18hr_05min.jpg|thumb|Gel 1]]
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[[Image:2006-08-11_18hr_03min.jpg|thumb|Gel 2]]
  
 
{| {{table}}
 
{| {{table}}
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* load 30 {{ul}} (of 50 {{ul}}) of supernatant into adjacent even-numbered lanes (e.g., trial 1-0 has pellet in lane 1 and supernatant in lane 2)
 
* load 30 {{ul}} (of 50 {{ul}}) of supernatant into adjacent even-numbered lanes (e.g., trial 1-0 has pellet in lane 1 and supernatant in lane 2)
 
* run at 60V for 1 h
 
* run at 60V for 1 h
 +
 +
Results/discussion
 +
* PEG precipitations appeared to have failed: no oligos were separated
 +
* curiously, the dye in the "supernatant" lanes ran at about 2/3 of the speed of the dye in the "pellet" lanes, and it gave a smear and not a band

Revision as of 15:03, 11 August 2006

Thoughts/ramblings/goals/questions/general frustrations

The results of yesterday's experiment show that Microcon filtration gives low yields and the PEG precipitation (at least at 10%) damages nanostructures regardless of folding conditions.

Questions

  • Are Microcon yields unacceptably low, or are they acceptable? (Can we use the NanoDrop to quantify our yield?)
    • Gels are much better for quantifying yield - you can try both and see how they compare.
  • Low concentrations of PEG should precipitate large nanostructures. Will some smaller concentration of PEG not harm nanostructures formed under some folding conditions?
    • Our August 2 experiment showed that even low concentrations of PEG damage nanostructures folded under "standard" conditions (10x oligos, 10 mM MgCl2).

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:IGEM060811-p7308a.jpg
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
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)

Gigundo PEG precipitation

  • goal: test 0% to 8% PEG precipitations with nanostructures folded under all six folding conditions from yesterday
  • optimistic hypothesis: nanostructures folded with higher concentrations of oligos and/or MgCl2 will show less damage after treatment with PEG

Protocol: prepare the following 30 samples.

Gel 1
Gel 2
Trial Final PEG % Gel Lanes 20% PEG (μL) 5 M NaCl (μL) Nanostructures (μL) water (μL) Total volume (μL)
1-0 0% 1 1 0 5 5 20 50
1-2 2% 1 3 5 5 5 15 50
1-4 4% 1 5 10 5 5 10 50
1-6 6% 1 7 15 5 5 5 50
1 kb+ ladder 1 9 10
p7308 1 10 10
1-8 8% 1 11 20 5 5 0 50
2-0 0% 1 13 0 5 5 20 50
2-2 2% 1 15 5 5 5 15 50
2-4 4% 1 17 10 5 5 10 50
2-6 6% 1 19 15 5 5 5 50
2-8 8% 1 21 20 5 5 0 50
3-0 0% 1 23 0 5 5 20 50
3-2 2% 1 25 5 5 5 15 50
3-4 4% 1 27 10 5 5 10 50
1 kb+ ladder 1 29 10
p7308 1 30 10
3-6 6% 1 31 15 5 5 5 50
3-8 8% 1 33 20 5 5 0 50
4-0 0% 1 35 0 5 5 20 50
4-2 2% 1 37 5 5 5 15 50
4-4 4% 1 39 10 5 5 10 50
4-6 6% 2 1 15 5 5 5 50
4-8 8% 2 3 20 5 5 0 50
5-0 0% 2 5 0 5 5 20 50
5-2 2% 2 7 5 5 5 15 50
1 kb+ ladder 1 29 10
p7308 1 30 10
5-4 4% 2 11 10 5 5 10 50
5-6 6% 2 13 15 5 5 5 50
5-8 8% 2 15 20 5 5 0 50
6-0 0% 2 17 0 5 5 20 50
6-2 2% 2 19 5 5 5 15 50
6-4 4% 2 21 10 5 5 10 50
6-6 6% 2 23 15 5 5 5 50
6-8 8% 2 25 20 5 5 0 50
1 kb+ ladder 1 29 10
p7308 1 30 10
  • incubate on ice for 15 min.
  • spin at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
  • carefully pipet off supernatant
  • resuspend "pellet" in 20 μL of respective folding buffer
  • load resuspended pellets in odd-numbered lanes of 2% TBE agarose gel supplemented to 10 mM MgCl2
  • load 30 μL (of 50 μL) of supernatant into adjacent even-numbered lanes (e.g., trial 1-0 has pellet in lane 1 and supernatant in lane 2)
  • run at 60V for 1 h

Results/discussion

  • PEG precipitations appeared to have failed: no oligos were separated
  • curiously, the dye in the "supernatant" lanes ran at about 2/3 of the speed of the dye in the "pellet" lanes, and it gave a smear and not a band