IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-11

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m (p7308 quantitation)
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| 8||p7308 060522, 1:2 dil (12 {{ul}})||AGLB (2 {{ul}})
| 8||p7308 060522, 1:2 dil (12 {{ul}})||AGLB (2 {{ul}})
|}
|}
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==Gigundo PEG precipitation==
 +
 +
* goal: test 0% to 8% PEG precipitations with nanostructures folded under all six folding conditions from yesterday
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* 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.
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 +
{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Trial'''
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| align="center" style="background:#f0f0f0;"|'''Final PEG %'''
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| align="center" style="background:#f0f0f0;"|'''20% PEG ({{ul}})'''
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| align="center" style="background:#f0f0f0;"|'''5 M NaCl ({{ul}})'''
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| align="center" style="background:#f0f0f0;"|'''Nanostructures ({{ul}})'''
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| align="center" style="background:#f0f0f0;"|'''water ({{ul}})'''
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| align="center" style="background:#f0f0f0;"|'''Total volume ({{ul}})'''
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|-
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| 1-0||0%||0||5||5||20||50
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|-
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| 1-2||2%||5||5||5||15||50
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|-
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| 1-4||4%||10||5||5||10||50
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|-
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| 1-6||6%||15||5||5||5||50
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|-
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| 1-8||8%||20||5||5||0||50
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|-
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| 2-0||0%||0||5||5||20||50
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|-
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| 2-2||2%||5||5||5||15||50
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|-
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| 2-4||4%||10||5||5||10||50
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|-
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| 2-6||6%||15||5||5||5||50
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|-
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| 2-8||8%||20||5||5||0||50
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|-
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| 3-0||0%||0||5||5||20||50
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|-
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| 3-2||2%||5||5||5||15||50
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|-
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| 3-4||4%||10||5||5||10||50
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|-
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| 3-6||6%||15||5||5||5||50
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|-
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| 3-8||8%||20||5||5||0||50
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|-
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| 4-0||0%||0||5||5||20||50
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|-
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| 4-2||2%||5||5||5||15||50
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|-
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| 4-4||4%||10||5||5||10||50
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|-
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| 4-6||6%||15||5||5||5||50
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|-
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| 4-8||8%||20||5||5||0||50
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|-
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| 5-0||0%||0||5||5||20||50
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|-
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| 5-2||2%||5||5||5||15||50
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|-
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| 5-4||4%||10||5||5||10||50
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|-
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| 5-6||6%||15||5||5||5||50
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|-
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| 5-8||8%||20||5||5||0||50
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|-
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| 6-0||0%||0||5||5||20||50
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|-
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| 6-2||2%||5||5||5||15||50
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|-
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| 6-4||4%||10||5||5||10||50
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|-
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| 6-6||6%||15||5||5||5||50
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|-
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| 6-8||8%||20||5||5||0||50
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|}
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* incubate on ice for 15 min.
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* spin at 16 k rcf at 4{{c}} for 10 min.
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* carefully pipet off supernatant
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* resuspend "pellet" in 10 {{ul}} of respective folding buffer

Revision as of 13:18, 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).
Image:IGEM060811-p7308a.jpg
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)

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.

Trial Final PEG % 20% PEG (μL) 5 M NaCl (μL) Nanostructures (μL) water (μL) Total volume (μL)
1-00%0552050
1-22%5551550
1-44%10551050
1-66%1555550
1-88%2055050
2-00%0552050
2-22%5551550
2-44%10551050
2-66%1555550
2-88%2055050
3-00%0552050
3-22%5551550
3-44%10551050
3-66%1555550
3-88%2055050
4-00%0552050
4-22%5551550
4-44%10551050
4-66%1555550
4-88%2055050
5-00%0552050
5-22%5551550
5-44%10551050
5-66%1555550
5-88%2055050
6-00%0552050
6-22%5551550
6-44%10551050
6-66%1555550
6-88%2055050
  • 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 10 μL of respective folding buffer
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