IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-4

PAGE of Concentrated Eb, Fb, and Gb

• given amounts of streptavidin were added to each component on parafilm
• 2uL of 10x loading dye was added to each mixture subsequently
• gel was run at 4°C for 1hr 10minutes at 100V
• gel was stained in coomassie blue from 12PM F 8/4/06 to ________

lane	component	concentration	amount of component in well μL	streptavidin added μL
1	streptavidin	2uM	3.5	-
2	p7308	44uM	7.5	3.5
3	c4.0.6b (ie. biotinylated inside-oligos)	50nM each oligo (as in all pre-working stocks)	7	3.5
4	Eb PEG-pellet	21/8 of a rxn	15	3.5
5	Fb PEG-pellet	21/8 of a rxn	15	3.5
6	Gb PEG-pellet	21/8 of a rxn	15	3.5
7	streptavidin	2uM	7	-

PEG precipitation w/ New Folded Rxns

• Goal: test PEG precipitation with folding rxns under new conditions
• Plan to use 50 μL final volume
• Add 10 μL 10 nM scaffold/100 nM oligo folded mix (since there was only 20 μL in total of each folded rxn)
• Add 20% PEG/2.5M NaCl solution (10 μL)
• Add as much water to each as it takes to get them to a 100 μL final volume (30 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 naked scaffold (1 lane)
  • Load 6 folding rxns untreated, supernatant, pellet (18 lanes)
  • 20 lanes total

Lane	Contents	Loading Dye
0	1kb DNA ladder (4 μL)	0 μL
1	naked scaffold (10 μL)	2 μL
2	10mM MgCl2 + 100nM oligos rxn untreated (10 μL)	2 μL
3	10mM MgCl2 + 100nM oligos rxn pellet (10 μL)	2 μL
4	10mM MgCl2 + 100nM oligos rxn supernatant (10 μL)	2 μL
5	20mM MgCl2 + 100nM oligos rxn untreated (10 μL)	2 μL
6	20mM MgCl2 + 100nM oligos rxn pellet (10 μL)	2 μL
7	20mM MgCl2 + 100nM oligos rxn supernatant (10 μL)	2 μL
8	30mM MgCl2 + 100nM oligos rxn untreated (10 μL)	2 μL
9	30mM MgCl2 + 100nM oligos rxn pellet (10 μL)	2 μL
10	30mM MgCl2 + 100nM oligos rxn supernatant (10 μL)	2 μL
11	10mM MgCl2 + 600nM oligos rxn untreated (10 μL)	2 μL
12	10mM MgCl2 + 600nM oligos rxn pellet (10 μL)	2 μL
13	10mM MgCl2 + 600nM oligos rxn supernatant (10 μL)	2 μL
14	20mM MgCl2 + 600nM oligos rxn untreated (10 μL)	2 μL
15	20mM MgCl2 + 600nM oligos rxn pellet (10 μL)	2 μL
16	20mM MgCl2 + 600nM oligos rxn supernatant (10 μL)	2 μL
17	30mM MgCl2 + 600nM oligos rxn untreated (10 μL)	2 μL
18	30mM MgCl2 + 600nM oligos rxn pellet (10 μL)	2 μL
19	30mM MgCl2 + 600nM oligos rxn supernatant (10 μL)	2 μL

• Results
  • no bands at all besides naked scaffold
  • have to redo 6 folding rxns