IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-1: Difference between revisions
(9 intermediate revisions by 3 users not shown) | |||
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==Ordering== | ==Ordering== | ||
[[Media: v5latches_v3-2attacholigs_v5attacholigs_orderform_TC.xls|Order form for v5.0 latches, v3.2 attachment oligos, and v5.0 attachment oligos]] | |||
* v5.0 latches and v3.2 and v5.0 ligand-attachment oligos ordered | * v5.0 latches and v3.2 and v5.0 ligand-attachment oligos ordered | ||
* forgot to order the ligands - will do tomorrow, along with the clarified v4.0 ligand-attachment oligos | * forgot to order the ligands - will do tomorrow, along with the clarified v4.0 ligand-attachment oligos | ||
Line 61: | Line 64: | ||
*goal: try again, different DNA | *goal: try again, different DNA | ||
*run on [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-2#SYBR_Gold_and_silver_staining|August 2]] | |||
==Container 5.0 lid refolding== | ==Container 5.0 lid refolding== | ||
Line 123: | Line 92: | ||
==PEG precipitation== | ==PEG precipitation== | ||
*Goal: repeat titration of PEG precipitation conditions for folded containers | *Goal: repeat titration of PEG precipitation conditions for folded containers | ||
**Katie and Val did one trial, Tiff and Matthew did another | |||
*'''Folding reactions''' | *'''Folding reactions''' | ||
**2 samples of 6hb (100 {{ul}} final volume) | **2 samples of 6hb (100 {{ul}} final volume) | ||
Line 138: | Line 108: | ||
*Add 20% PEG solution (40 {{ul}}, 60 {{ul}}, 80 {{ul}}, 100 {{ul}}, 120 {{ul}}, 140 {{ul}}) | *Add 20% PEG solution (40 {{ul}}, 60 {{ul}}, 80 {{ul}}, 100 {{ul}}, 120 {{ul}}, 140 {{ul}}) | ||
*Add 5 M NaCl stock solution (20 {{ul}}) | *Add 5 M NaCl stock solution (20 {{ul}}) | ||
*Add as much water to each as it takes to get them to a 200 {{ul}} final volume | |||
*Incubate on ice for 15 minutes | *Incubate on ice for 15 minutes | ||
*Spin 16k rcf for 10 minutes | *Spin 16k rcf for 10 minutes | ||
** ''0.2 mL PCR tubes broke through the 1.5 mL microcentrifuge tubes that had been holding them. Will try again using 1.5 mL PCR tubes for reaction mixtures.''' | ** ''Tiff and Matt's trial: 0.2 mL PCR tubes broke through the 1.5 mL microcentrifuge tubes that had been holding them. Will try again using 1.5 mL PCR tubes for reaction mixtures.''' | ||
*Pipette out supernatant into separate tube | *Pipette out supernatant into separate tube | ||
*Resuspend pellet in 1x folding buffer volume equal to the supernatant | *Resuspend pellet in 1x folding buffer volume equal to the supernatant | ||
Line 152: | Line 123: | ||
**19 lanes total | **19 lanes total | ||
Katie and Val's results: | |||
[[Image:IGEM06-SD-0600801b.jpg|thumb|2% agarose gel, 0.5 mg/mL EtBr<br>0.5x TBE, 11 mM {{mgcl2}}]] | [[Image:IGEM06-SD-0600801b.jpg|thumb|2% agarose gel, 0.5 mg/mL EtBr<br>0.5x TBE, 11 mM {{mgcl2}}]] | ||
{| {{table}} | {| {{table}} | ||
Line 196: | Line 168: | ||
| 18||c5 barrel 14% PEG pellet (10 {{ul}})||AGLB (2 {{ul}}) | | 18||c5 barrel 14% PEG pellet (10 {{ul}})||AGLB (2 {{ul}}) | ||
|} | |} | ||
[[Image:IGEM06-SD-0600801c.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||[[:Image:1kb.png|1kb DNA ladder]] (4 {{ul}})|| | |||
|- | |||
| 1||c5 barrel untreated (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 2||c5 barrel 4% PEG supernatant (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 3||c5 barrel 4% PEG pellet (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 4||c5 barrel 5% PEG supernatant (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 5||c5 barrel 5% PEG pellet (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 6||c5 barrel 6% PEG supernatant (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 7||c5 barrel 6% PEG pellet (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 8||c5 barrel 10% PEG supernatant (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 9||c5 barrel 10% PEG pellet (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 10||c5 barrel 14% PEG supernatant (10 {{ul}})||AGLB (2 {{ul}}) | |||
|- | |||
| 11||c5 barrel 14% PEG pellet (10 {{ul}})||AGLB (2 {{ul}}) | |||
|} | |||
==EM imaging== | |||
<gallery> | |||
Image:IGEM06-EM0801-1a.jpg|c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-1b.jpg|c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-1c.jpg|c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-1d.jpg|c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-1e.jpg|c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-1f.jpg|c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-2a.jpg|c5.0 lid2 (2 nM), 6hb (0.4 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-2b.jpg|c5.0 lid2 (2 nM), 6hb (0.4 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-2c.jpg|c5.0 lid2 (2 nM), 6hb (0.4 nM), 0.2% uranyl formate | |||
Image:IGEM06-EM0801-2d.jpg|c5.0 lid2 (2 nM), 6hb (0.4 nM), 0.2% uranyl formate | |||
</gallery> | |||
==High Concentrations of Nanoboxes, Day 2== | |||
* Qiagen gel purified 3rxns of each Eb and Gb. Used 2% agarose Mg2+-supplemented gel. Eluted each column (used 2/3rxns, so as to try to avoid overloading the column filter) into 40uL of EB, leaving 3rxns worth of nanoboxes (approx 200*3fmol, or 600fmol of biotin binding sites) in 80uL of solution. | |||
** scaffold (2nd lane) and folded Eb and Gb (3rd-14th lane) run differently - indicates folding worked | |||
<gallery> | |||
Image:Eb_Gb_folding_TC_8_1_2006.jpg|2% agarose Mg2+ gel with Eb (six lanes after first two) and Gb (six lanes after Eb's six)]] | |||
</gallery> | |||
* PEG precipitated at 12% total concentrations. Accidentally tossed out the supernatant. Reconstituted pellet in 80ul of 1x folding buffer, to try to get comparable concentrations of nanoboxes. | |||
* Ran 10ul of each reaction on 2% agarose Mg2+-supplemented gel (wanted to run with scaffold, but none left in the lab). Saw bright bands for PEG-precipitated boxes, none for gel-purified. Mg2+ in the original gel might have interfered with column interaction. | |||
** gel purification shows nothing - didn't work, or concentrations too low | |||
***'''NB: Shawn has said that the Qiagen gel purification reagents will cause unfolding of the boxes - gel purification should therefore no longer be used for nanoboxes.''' | |||
**PEG-pelleted nanoboxes look bright (even though this is only 10uL of a 3-rxn condensation - in essence, 3/8 of a normal 40uL-rxn) | |||
<gallery> | |||
Image:gel_PEG_purify_Eb_Gb_TC_8_1_2006.jpg|2% agarose Mg2+ gel with gel purified Eb (2nd lane), Gb (3rd lane), and PEG-pellets of Eb (4th lane), Gb (5th lane)]] | |||
</gallery> |
Latest revision as of 14:48, 3 August 2006
To do today
- ordering
- new oligo ligands
- AscIII (if not already ordered
- PEG precipitations
- repeat experiment, run on PA gel, run on agarose gel
- SYBR gold
- read technical specs
- determine lower threshold of imaging
Ordering
Order form for v5.0 latches, v3.2 attachment oligos, and v5.0 attachment oligos
- v5.0 latches and v3.2 and v5.0 ligand-attachment oligos ordered
- forgot to order the ligands - will do tomorrow, along with the clarified v4.0 ligand-attachment oligos
SYBR gold testing
Trial 1
- goal: to determine the minimum amount of DNA that can be visualized with SYBR Gold
- methods: 12% PA gel with lanes listed below, run for 30 min. at 130 V, then stained with Invitrogen SYBR Gold.
- prepared 1x solution: 10 μL thawed 10,000x SYBR gold in 100 mL TBE, stored in light-proof container at 4[[:Category:{{{1}}}|{{{1}}}]].
lane | DNA (3.2.7.2b) (12.5 pg DNA / fmol) | ||
(μL) | (fmols) | (pg) | |
1 | 15 μL 1 μM | 15,000 | 187,500 |
2 | 10 μL 1 μM | 10,000 | 125,000 |
3 | 5 μL 1 μM | 5,000 | 62,500 |
4 | 2.5 μL 1.0 μM | 2,500 | 31,250 |
5 | 1 μL 1.0 μM | 1,000 | 12,500 |
6 | 5 μL 0.1 μM | 500 | 6,250 |
7 | 2.5 μL 0.1 μM | 250 | 3,125 |
8 | 1 μL 0.1 μM | 100 | 1,250 |
9 | 5 μL 0.01 μM | 50 | 625 |
10 | 2.5 μL 0.01 μM | 25 | 313 |
11 | 1 μL 0.01 μM | 10 | 125 |
12 | 10 μL 1kb+ ladder |
- stained with 1x SYBR Gold for 50 min.
- results
- ladder stained brilliantly, visible both under SYBR Gold filter (and less so under EtBr filter)
- no other DNAs showed, including after EtBr soak
- dye was only halfway down gel, so ss oligos probably didn't run off
- will try again with non biotinylated oligo
Trial 2
- goal: try again, different DNA
- run on August 2
Container 5.0 lid refolding
- Yesterday's gel result indicated that folding two lids from a single scaffold didn't work well
- Goal: fold c5.0 lids using separate scaffolds
Lane | Contents | Loading Buffer |
0 | 1kb DNA ladder (4 μL) | |
1 | naked p7704 (10 μL) | AGLB (2 μL) |
2 | c5.0 lid 1 (10 μL) | AGLB (2 μL) |
3 | c5.0 lid 2 (10 μL) | AGLB (2 μL) |
4 | c5.0 lid 1+2 (10 μL) | AGLB (2 μL) |
- Lid 1 seems to be causing the smearing
- Lid 1+2 looks a lot better in this trial for some reason
- Proper p7572 scaffold may improve things further
PEG precipitation
- Goal: repeat titration of PEG precipitation conditions for folded containers
- Katie and Val did one trial, Tiff and Matthew did another
- Folding reactions
- 2 samples of 6hb (100 μL final volume)
- 4 samples of design 5 barrels (100 μL volume)
- Mix the following
- 10 uL 500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2
- 40 uL working stock 250 nM each oligo (100 nM each oligo final concentration)
- 50 uL p7308 20 nM (10 nM final concentration)
- Anneal from 80[[:Category:{{{1}}}|{{{1}}}]] to 20[[:Category:{{{1}}}|{{{1}}}]], -1[[:Category:{{{1}}}|{{{1}}}]] per min
- PEG precipitations
- Plan to use 200 μL final volume
- Trying 4%, 6%, 8%, 10%, 12%, 14%
- Add 40 μL 10 nM scaffold/100 nM oligo folded mix
- Add 20% PEG solution (40 μL, 60 μL, 80 μL, 100 μL, 120 μL, 140 μL)
- Add 5 M NaCl stock solution (20 μL)
- Add as much water to each as it takes to get them to a 200 μL final volume
- Incubate on ice for 15 minutes
- Spin 16k rcf for 10 minutes
- Tiff and Matt's trial: 0.2 mL PCR tubes broke through the 1.5 mL microcentrifuge tubes that had been holding them. Will try again using 1.5 mL PCR tubes for reaction mixtures.'
- Pipette out supernatant into separate tube
- Resuspend pellet in 1x folding buffer volume equal to the supernatant
- Run equivalent volumes of pellet and sup on 2% agarose gel (+ 11 mM MgCl2)
- Gel Analysis
- Analyze on 2% Mg agarose gel (0.5x TBE, 11 mM MgCl2, 0.5 μg/mL EtBr)
- Load 1kb ladder (1 lane)
- Load c5 supernatant, pellet for 0%, 4%, 6%, 8%, 10%, 12%, 14% (13 lanes)
- Load 6hb supernatant, pellet for 0%, 4%, 14% (5 lanes)
- 19 lanes total
Katie and Val's results:
Lane | Contents | Loading Buffer |
0 | 1kb DNA ladder (4 μL) | |
1 | 6hb untreated (10 μL) | AGLB (2 μL) |
2 | 6hb 4% PEG supernatant (10 μL) | AGLB (2 μL) |
3 | 6hb 4% PEG pellet (10 μL) | AGLB (2 μL) |
4 | 6hb 14% PEG supernatant (10 μL) | AGLB (2 μL) |
5 | 6hb 14% PEG pellet (10 μL) | AGLB (2 μL) |
6 | c5 barrel untreated (10 μL) | AGLB (2 μL) |
7 | c5 barrel 4% PEG supernatant (10 μL) | AGLB (2 μL) |
8 | c5 barrel 4% PEG pellet (10 μL) | AGLB (2 μL) |
9 | c5 barrel 6% PEG supernatant (10 μL) | AGLB (2 μL) |
10 | c5 barrel 6% PEG pellet (10 μL) | AGLB (2 μL) |
11 | c5 barrel 8% PEG supernatant (10 μL) | AGLB (2 μL) |
12 | c5 barrel 8% PEG pellet (10 μL) | AGLB (2 μL) |
13 | c5 barrel 10% PEG supernatant (10 μL) | AGLB (2 μL) |
14 | c5 barrel 10% PEG pellet (10 μL) | AGLB (2 μL) |
15 | c5 barrel 12% PEG supernatant (10 μL) | AGLB (2 μL) |
16 | c5 barrel 12% PEG pellet (10 μL) | AGLB (2 μL) |
17 | c5 barrel 14% PEG supernatant (10 μL) | AGLB (2 μL) |
18 | c5 barrel 14% PEG pellet (10 μL) | AGLB (2 μL) |
Lane | Contents | Loading Buffer |
0 | 1kb DNA ladder (4 μL) | |
1 | c5 barrel untreated (10 μL) | AGLB (2 μL) |
2 | c5 barrel 4% PEG supernatant (10 μL) | AGLB (2 μL) |
3 | c5 barrel 4% PEG pellet (10 μL) | AGLB (2 μL) |
4 | c5 barrel 5% PEG supernatant (10 μL) | AGLB (2 μL) |
5 | c5 barrel 5% PEG pellet (10 μL) | AGLB (2 μL) |
6 | c5 barrel 6% PEG supernatant (10 μL) | AGLB (2 μL) |
7 | c5 barrel 6% PEG pellet (10 μL) | AGLB (2 μL) |
8 | c5 barrel 10% PEG supernatant (10 μL) | AGLB (2 μL) |
9 | c5 barrel 10% PEG pellet (10 μL) | AGLB (2 μL) |
10 | c5 barrel 14% PEG supernatant (10 μL) | AGLB (2 μL) |
11 | c5 barrel 14% PEG pellet (10 μL) | AGLB (2 μL) |
EM imaging
-
c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate
-
c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate
-
c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate
-
c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate
-
c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate
-
c5.0 barrel (3.3 nM), 6hb (3.3 nM), 0.2% uranyl formate
-
c5.0 lid2 (2 nM), 6hb (0.4 nM), 0.2% uranyl formate
-
c5.0 lid2 (2 nM), 6hb (0.4 nM), 0.2% uranyl formate
-
c5.0 lid2 (2 nM), 6hb (0.4 nM), 0.2% uranyl formate
-
c5.0 lid2 (2 nM), 6hb (0.4 nM), 0.2% uranyl formate
High Concentrations of Nanoboxes, Day 2
- Qiagen gel purified 3rxns of each Eb and Gb. Used 2% agarose Mg2+-supplemented gel. Eluted each column (used 2/3rxns, so as to try to avoid overloading the column filter) into 40uL of EB, leaving 3rxns worth of nanoboxes (approx 200*3fmol, or 600fmol of biotin binding sites) in 80uL of solution.
- scaffold (2nd lane) and folded Eb and Gb (3rd-14th lane) run differently - indicates folding worked
-
2% agarose Mg2+ gel with Eb (six lanes after first two) and Gb (six lanes after Eb's six)]]
- PEG precipitated at 12% total concentrations. Accidentally tossed out the supernatant. Reconstituted pellet in 80ul of 1x folding buffer, to try to get comparable concentrations of nanoboxes.
- Ran 10ul of each reaction on 2% agarose Mg2+-supplemented gel (wanted to run with scaffold, but none left in the lab). Saw bright bands for PEG-precipitated boxes, none for gel-purified. Mg2+ in the original gel might have interfered with column interaction.
- gel purification shows nothing - didn't work, or concentrations too low
- NB: Shawn has said that the Qiagen gel purification reagents will cause unfolding of the boxes - gel purification should therefore no longer be used for nanoboxes.
- PEG-pelleted nanoboxes look bright (even though this is only 10uL of a 3-rxn condensation - in essence, 3/8 of a normal 40uL-rxn)
- gel purification shows nothing - didn't work, or concentrations too low
-
2% agarose Mg2+ gel with gel purified Eb (2nd lane), Gb (3rd lane), and PEG-pellets of Eb (4th lane), Gb (5th lane)]]