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

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(Gigundo PEG precipitation)
Line 59: Line 59:
| 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 67:
| 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 147:
* 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

Revision as of 17:43, 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 % 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
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