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

SYBR gold testing

• 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 20 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	2 μL 1kb+ ladder

PEG precipitation

• Goal: repeat titration of PEG precipitation conditions for folded containers
• 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)
• Incubate on ice for 15 minutes
• Spin 16k rcf for 10 minutes
• 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

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)