IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-1

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

2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl₂

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:

2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl₂

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)

File:IGEM06-SD-0600801c.jpg

2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl₂

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)

• 

  2% agarose Mg2+ gel with gel purified Eb (2nd lane), Gb (3rd lane), and PEG-pellets of Eb (4th lane), Gb (5th lane)]]