IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-9

Folding reaction

Fold 200 μL each of 3.2.Fo, .Go, .Ho, .Io

Each reaction:

• 45 μL p7308 scaffold (44 nM)
• 80 μL oligo stock (250 nM)
• 20 μL 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl₂)
• 55 μL dH₂O

Folding conditions:

• high-volume protocol: start at 80[[:Category:{{{1}}}|{{{1}}}]], decrement 0.5[[:Category:{{{1}}}|{{{1}}}]] every 1 min. for 120 cycles.

Gel analysis

• see below

More Microcon trials

Another protocol, inspired by Millipore's Concentrating and Desalting DNA or RNA with Microcon or Centricon Centrifugal Filters:

• 20 μL folded nanostructures (4.0) into a YM-50 Microcon tube
• 480 μL water added
• centrifuged at 14,000 g for 6 min.
• flow-through discarded
• centrifuged upside-down at 1,300 g for 3 min. to collect retentate
  • 53 μL collected

Gel analysis

• ran half of retentate, along with half of starting nanostructure volume for comparison
• also ran 5 μL of each nanostructure folded today to see if they folded properly

Lane	Contents
1	10 μL nanostructures
2	26.5 μL retentate
3	1 kb+ ladder
4	p7308
5	5 μL 3.2.Fo
6	5 μL 3.2.Go
7	5 μL 3.2.Ho
8	5 μL 3.2.Io

• results/discussion
  • Microcon yield is reasonably good, but lots of oligos remain
  • in the future: go back to repeating wash four times for 6 min each time (the maximum number of washes, according to Microcon)
  • folding reactions appear to have folded properly

Microcon purification of yesterday's folding reactions

• speedvac'd c3.2F, G, H, I, Eb, Fb flowthrough for 50 min. (vol. def much higher than 40 μL)
• ran 2% agarose gel + MgCl₂
• 3 flowthroughs is probably not enough - notice pretty clear oligo smears: should probably do 5/6
• don't know if oligo-ligand and latches were added to rxns before microcon: repeat folding of c3.2F, G, H, I and retest

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

Lane	Contents	Loading Buffer
0	1kb ladder (10 μL)	-
1	naked p7308 (7.5 μL)	5 μL
2	c3.2F (7.5 μL)	5 μL
3	c3.2F flowthrough (35 μL)	5 μL
4	c3.2G (7.5 μL)	5 μL
5	c3.2G flowthrough (35 μL)	5 μL
6	c3.2H (7.5 μL)	5 μL
7	c3.2H flowthrough (35 μL)	5 μL
8	c3.2I (7.5 μL)	5 μL
9	c3.2I flowthrough (35 μL)	5 μL
10	c5.0Eb (7.5 μL)	5 μL
11	c5.0Eb flowthrough (35 μL)	5 μL
12	c5.0Fb (7.5 μL)	5 μL
13	c5.0Fb flowthrough (35 μL)	5 μL