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

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Current revision (15:22, 15 August 2006) (view source)
(p7308 quantitation)
 
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** Our [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-2#PEG_precipitation|August 2 experiment]] showed that even low concentrations of PEG damage nanostructures folded under "standard" conditions (10x oligos, 10 mM {{mgcl2}}).
** Our [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-2#PEG_precipitation|August 2 experiment]] showed that even low concentrations of PEG damage nanostructures folded under "standard" conditions (10x oligos, 10 mM {{mgcl2}}).
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==p7308 quantitation==
 
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*Speedvac 060522 p7308 sample down to 50% volume.  This should remove any ethanol, and give you a slightly more manageable volume.
 
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*Pour 2% agraose, 11 mM {{mgcl2}} gel
 
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*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.
 
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*Load gel according to table below
 
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*Run for 2 hrs, 70V
 
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*When imaging gel, use spot density tool to measure intensity of each band
 
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**Use saturation indicator to take a picture just below the point where any bands start saturating on the image
 
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**Draw a rectangle that fits around the largest band on the gel
 
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**Copy that rectangle and position it directly above the first band.  This will be used to measure background
 
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**Repeat this for every band on the gel (one box for the band, one box for background)
 
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**Record this data along with gel picture on the wiki
 
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*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
 
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**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).
 
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[[Image:IGEM060811-p7308a.jpg|thumb|2% agarose gel, 0.5 mg/mL EtBr<br>0.5x TBE, 11 mM {{mgcl2}}]]
 
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{| {{table}}
 
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| align="center" style="background:#f0f0f0;"|'''Lane'''
 
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| align="center" style="background:#f0f0f0;"|'''Contents'''
 
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| align="center" style="background:#f0f0f0;"|'''Loading Buffer'''
 
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|-
 
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| 0||1kb DNA ladder (5 {{ul}})||
 
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|-
 
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| 1||p7308 060323, 44 nM (3 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (9 {{ul}})
 
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|-
 
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| 2||p7308 060323, 44 nM (6 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (6 {{ul}})
 
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|-
 
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| 3||p7308 060323, 44 nM (9 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (3 {{ul}})
 
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|-
 
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| 4||p7308 060522, 1:2 dil (1 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (11 {{ul}})
 
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|-
 
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| 5||p7308 060522, 1:2 dil (3 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (9 {{ul}})
 
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|-
 
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| 6||p7308 060522, 1:2 dil (6 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (6 {{ul}})
 
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|-
 
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| 7||p7308 060522, 1:2 dil (9 {{ul}})||AGLB (2 {{ul}}) + d{{h2o}} (3 {{ul}})
 
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|-
 
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| 8||p7308 060522, 1:2 dil (12 {{ul}})||AGLB (2 {{ul}})
 
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|}
 
==Gigundo PEG precipitation==
==Gigundo PEG precipitation==

Current revision

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).


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 1
Gel 2
Gel 2
Trial Final PEG % Gel Lanes 20% PEG (μL) 5 M NaCl (μL) Nanostructures (μL) water (μL) Total volume (μL)
1-00%110552050
1-22%135551550
1-44%1510551050
1-66%171555550
1 kb+ ladder1910
p730811010
1-88%1112055050
2-00%1130552050
2-22%1155551550
2-44%11710551050
2-66%1191555550
2-88%1212055050
3-00%1230552050
3-22%1255551550
3-44%12710551050
1 kb+ ladder12910
p730813010
3-66%1311555550
3-88%1332055050
4-00%1350552050
4-22%1375551550
4-44%13910551050
4-66%211555550
4-88%232055050
5-00%250552050
5-22%275551550
1 kb+ ladder12910
p730813010
5-44%21110551050
5-66%2131555550
5-88%2152055050
6-00%2170552050
6-22%2195551550
6-44%22110551050
6-66%2231555550
6-88%2252055050
1 kb+ ladder12910
p730813010
  • 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

Incubation of 3.2.E with thrombin beads

  • Goal: test if we can detect the binding of a nanostructure with outside aptamers to thrombin beads.

100 uL 10 nM 3.2.E or 100 uL 10 nM mix of 6.4. H/I was incubated with 250 uL thrombin beads (supplied as a 50% slurry). 3.2.E has outside aptamer sequences while 6.4.H/I do not. Following a 30 minute incubation, the beads were washed. The beads were then eluted by incubating with 250 uL 50% w/v free thrombin for 30 minutes. Washes and elutions were run on a 2% agarose gel for 60 minutes at 60V.

Lane Contents
01kb+ DNA ladder
26.4. H/I wash
46.4. H/I elution
63.2.E wash
83.2.E elution

Image:nano811beadgel.jpg

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