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

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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}}).


==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).


[[Image:IGEM060811-p7308a.jpg|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}})
|}


==Gigundo PEG precipitation==
==Gigundo PEG precipitation==
Line 55: Line 17:


Protocol: prepare the following 30 samples.
Protocol: prepare the following 30 samples.
[[Image:2006-08-11_18hr_03min.jpg|thumb|Gel 1]]
[[Image:2006-08-11_18hr_05min.jpg|thumb|Gel 2]]


{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Trial'''
| align="center" style="background:#f0f0f0;"|'''Trial'''
| align="center" style="background:#f0f0f0;"|'''Final PEG %'''
| align="center" style="background:#f0f0f0;"|'''Final PEG %'''
| align="center" style="background:#f0f0f0;"|'''Gel'''
| align="center" style="background:#f0f0f0;"|'''Lanes'''
| align="center" style="background:#f0f0f0;"|'''20% PEG ({{ul}})'''
| align="center" style="background:#f0f0f0;"|'''20% PEG ({{ul}})'''
| align="center" style="background:#f0f0f0;"|'''5 M NaCl ({{ul}})'''
| align="center" style="background:#f0f0f0;"|'''5 M NaCl ({{ul}})'''
Line 65: Line 32:
| align="center" style="background:#f0f0f0;"|'''Total volume ({{ul}})'''
| align="center" style="background:#f0f0f0;"|'''Total volume ({{ul}})'''
|-
|-
| 1-0||0%||0||5||5||20||50
| 1-0||0%||1||1||0||5||5||20||50
|-
|-
| 1-2||2%||5||5||5||15||50
| 1-2||2%||1||3||5||5||5||15||50
|-
|-
| 1-4||4%||10||5||5||10||50
| 1-4||4%||1||5||10||5||5||10||50
|-
|-
| 1-6||6%||15||5||5||5||50
| 1-6||6%||1||7||15||5||5||5||50
|-
|-
| 1-8||8%||20||5||5||0||50
| 1 kb+ ladder||||1||9||||||||||10
|-
|-
| 2-0||0%||0||5||5||20||50
| p7308||||1||10||||||||||10
|-
|-
| 2-2||2%||5||5||5||15||50
| 1-8||8%||1||11||20||5||5||0||50
|-
|-
| 2-4||4%||10||5||5||10||50
| 2-0||0%||1||13||0||5||5||20||50
|-
|-
| 2-6||6%||15||5||5||5||50
| 2-2||2%||1||15||5||5||5||15||50
|-
|-
| 2-8||8%||20||5||5||0||50
| 2-4||4%||1||17||10||5||5||10||50
|-
|-
| 3-0||0%||0||5||5||20||50
| 2-6||6%||1||19||15||5||5||5||50
|-
|-
| 3-2||2%||5||5||5||15||50
| 2-8||8%||1||21||20||5||5||0||50
|-
|-
| 3-4||4%||10||5||5||10||50
| 3-0||0%||1||23||0||5||5||20||50
|-
|-
| 3-6||6%||15||5||5||5||50
| 3-2||2%||1||25||5||5||5||15||50
|-
|-
| 3-8||8%||20||5||5||0||50
| 3-4||4%||1||27||10||5||5||10||50
|-
|-
| 4-0||0%||0||5||5||20||50
| 1 kb+ ladder||||1||29||||||||||10
|-
|-
| 4-2||2%||5||5||5||15||50
| p7308||||1||30||||||||||10
|-
|-
| 4-4||4%||10||5||5||10||50
| 3-6||6%||1||31||15||5||5||5||50
|-
|-
| 4-6||6%||15||5||5||5||50
| 3-8||8%||1||33||20||5||5||0||50
|-
|-
| 4-8||8%||20||5||5||0||50
| 4-0||0%||1||35||0||5||5||20||50
|-
|-
| 5-0||0%||0||5||5||20||50
| 4-2||2%||1||37||5||5||5||15||50
|-
|-
| 5-2||2%||5||5||5||15||50
| 4-4||4%||1||39||10||5||5||10||50
|-
|-
| 5-4||4%||10||5||5||10||50
| 4-6||6%||2||1||15||5||5||5||50
|-
|-
| 5-6||6%||15||5||5||5||50
| 4-8||8%||2||3||20||5||5||0||50
|-
|-
| 5-8||8%||20||5||5||0||50
| 5-0||0%||2||5||0||5||5||20||50
|-
|-
| 6-0||0%||0||5||5||20||50
| 5-2||2%||2||7||5||5||5||15||50
|-
|-
| 6-2||2%||5||5||5||15||50
| 1 kb+ ladder||||1||29||||||||||10
|-
|-
| 6-4||4%||10||5||5||10||50
| p7308||||1||30||||||||||10
|-
|-
| 6-6||6%||15||5||5||5||50
| 5-4||4%||2||11||10||5||5||10||50
|-
|-
| 6-8||8%||20||5||5||0||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
|}
|}


Line 129: Line 112:
* spin at 16 k rcf at 4{{c}} for 10 min.
* spin at 16 k rcf at 4{{c}} for 10 min.
* carefully pipet off supernatant
* carefully pipet off supernatant
* resuspend "pellet" in 10 {{ul}} of respective folding buffer
* resuspend "pellet" in 20 {{ul}} of respective folding buffer
* load resuspended pellets in odd-numbered lanes of 2% TBE agarose gel supplemented to 10 mM {{mgcl2}}
* 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
 
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.
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Contents'''
|-
| 0||1kb+ DNA ladder
|-
| 2||6.4. H/I wash
|-
| 4||6.4. H/I elution
|-
| 6||3.2.E wash
|-
| 8||3.2.E elution
|}
 
[[Image:nano811beadgel.jpg]]

Latest revision as of 13:22, 15 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).


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

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
0 1kb+ DNA ladder
2 6.4. H/I wash
4 6.4. H/I elution
6 3.2.E wash
8 3.2.E elution

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