IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-1: Difference between revisions
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==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) | ||
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*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 | ||
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**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}} |
Revision as of 08:03, 2 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
- 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
lane | DNA (3.2.8.1) (13.9 pg DNA / fmol) |
water (μL) | ||
(μL) | (fmols) | (pg) | ||
1 | 15 μL 1 μM | 15,000 | 209,000 | |
2 | 10 μL 1 μM | 10,000 | 139,000 | |
3 | 5 μL 1 μM | 5,000 | 69,500 | |
4 | 2.5 μL 1.0 μM | 2,500 | 34,800 | |
5 | 1 μL 1.0 μM | 1,000 | 13,900 | |
6 | 5 μL 0.1 μM | 500 | 6,950 | |
7 | 2.5 μL 0.1 μM | 250 | 3,480 | |
8 | 1 μL 0.1 μM | 100 | 1,390 | |
9 | 5 μL 0.01 μM | 50 | 695 | |
10 | 2.5 μL 0.01 μM | 25 | 348 | |
11 | 1 μL 0.01 μM | 10 | 139 | |
12 | 2 μL 1kb+ ladder |
- loaded in two gels
- ran at 130V for 30 min.
- stained on in silver stain, the other in SYBR gold
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 3% PEG supernatant (10 μL) | AGLB (2 μL) |
3 | c5 barrel 3% PEG pellet (10 μL) | AGLB (2 μL) |
4 | c5 barrel 4% PEG supernatant (10 μL) | AGLB (2 μL) |
5 | c5 barrel 4% PEG pellet (10 μL) | AGLB (2 μL) |
6 | c5 barrel 5% PEG supernatant (10 μL) | AGLB (2 μL) |
7 | c5 barrel 5% PEG pellet (10 μL) | AGLB (2 μL) |
8 | c5 barrel 6% PEG supernatant (10 μL) | AGLB (2 μL) |
9 | c5 barrel 6% PEG pellet (10 μL) | AGLB (2 μL) |