IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-17

2nd PEG Precipitation

• conduct 2nd PEG precipitation on yesterday's precipitated samples (5.0A, C, D 7%-10%)
• protocol: mix all ingredients, add nanostructures last, total volume is 100 mL, incubate on ice for 15 min, spin for 10 min at 16k rcf, pipet off supernatant, resuspend pellet in 100 μL water
• CONCLUSIONS: we seem to be unable to perform 2 PEG precipitations because all of the nanostructures ended up in the supernatant. we will repeat the 1st PEG precipitation with c5.0 A, C, D at 6% and probably not repeat another PEG if the protection assay is okay.

Final PEG %	PEG μL	NaCl μL	c5.0 μL	dH2O μL
7	35	10	40	15
8	40	10	40	10
9	45	10	40	5
10	50	10	40	0

lane	contents	loading dye
1	8 μL untreated 5.0.A	3 μL
2	20 μL 5.0.A.1 in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
3	8 μL 5.0.A.1 in 7% PEG and 0.5 M NaCl (pellet)	3 μL
4	20 μL 5.0.A.2 in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
5	8 μL 5.0.A.2 in 7% PEG and 0.5 M NaCl (pellet)	3 μL
6	20 μL 5.0.A.1 in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
7	8 μL 5.0.A.1 in 8% PEG and 0.5 M NaCl (pellet)	3 μL
8	20 μL 5.0.A.2 in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
9	8 μL 5.0.A.2 in 8% PEG and 0.5 M NaCl (pellet)	3 μL
10	1 kb+ ladder	3 μL
11	2.7 μL p7308	3 μL
12	20 μL 5.0.A.1 in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
13	8 μL 5.0.A.1 in 9% PEG and 0.5 M NaCl (pellet)	3 μL
14	20 μL 5.0.A.2 in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
15	8 μL 5.0.A.2 in 9% PEG and 0.5 M NaCl (pellet)	3 μL
16	20 μL 5.0.A.1 in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
17	8 μL 5.0.A.1 in 10% PEG and 0.5 M NaCl (pellet)	3 μL
18	20 μL 5.0.A.2 in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
19	8 μL 5.0.A.2 in 10% PEG and 0.5 M NaCl (pellet)	3 μL
20	empty	-
21	8 μL untreated 5.0.C	3 μL
22	20 μL 5.0.C.1 in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
23	8 μL 5.0.C.1 in 7% PEG and 0.5 M NaCl (pellet)	3 μL
24	20 μL 5.0.C.2 in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
23	8 μL 5.0.C.2 in 7% PEG and 0.5 M NaCl (pellet)	3 μL
24	20 μL 5.0.C.1 in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
25	8 μL 5.0.C.1 in 8% PEG and 0.5 M NaCl (pellet)	3 μL
26	20 μL 5.0.C.2 in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
27	8 μL 5.0.C.2 in 8% PEG and 0.5 M NaCl (pellet)	3 μL
28	20 μL 5.0.C.1 in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
29	8 μL 5.0.C.1 in 9% PEG and 0.5 M NaCl (pellet)	3 μL
30	1 kb+ ladder	3 μL
31	2.7 μL p7308	3 μL
32	20 μL 5.0.C.2 in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
33	8 μL 5.0.C.2 in 9% PEG and 0.5 M NaCl (pellet)	3 μL
34	20 μL 5.0.C.1 in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
35	8 μL 5.0.C.1 in 10% PEG and 0.5 M NaCl (pellet)	3 μL
36	20 μL 5.0.C.2 in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
37	8 μL 5.0.C.2 in 10% PEG and 0.5 M NaCl (pellet)	3 μL
38-40	empty	-
41	8 μL untreated 5.0.D	3 μL
42	20 μL 5.0.D.1 in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
43	8 μL 5.0.D.1 in 7% PEG and 0.5 M NaCl (pellet)	3 μL
44	20 μL 5.0.D.2 in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
45	8 μL 5.0.D.2 in 7% PEG and 0.5 M NaCl (pellet)	3 μL
46	20 μL 5.0.D.1 in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
47	8 μL 5.0.D.1 in 8% PEG and 0.5 M NaCl (pellet)	3 μL
48	20 μL 5.0.D.2 in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
49	8 μL 5.0.D.2 in 8% PEG and 0.5 M NaCl (pellet)	3 μL
50	1 kb+ ladder	3 μL
51	2.7 μL p7308	3 μL
52	20 μL 5.0.D.1 in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
53	8 μL 5.0.D.1 in 9% PEG and 0.5 M NaCl (pellet)	3 μL
54	20 μL 5.0.D.2 in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
55	8 μL 5.0.D.2 in 9% PEG and 0.5 M NaCl (pellet)	3 μL
56	20 μL 5.0.D.1 in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
57	8 μL 5.0.D.1 in 10% PEG and 0.5 M NaCl (pellet)	3 μL
58	20 μL 5.0.D.2 in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
59	8 μL 5.0.D.2 in 10% PEG and 0.5 M NaCl (pellet)	3 μL
60	empty	-

Magnetic Streptavidin Protection, Take 3

• Performed steps 9-12, as in Wed's protocol.

• Mistakes:
  • Speedvaced the final-supernatants for 1hr at 30[[:Category:{{{1}}}|{{{1}}}]], saw that about 90uL remained from the 200uL started with. Thought it would be alright to let it continue Speedvacing for another 30 minutes, as the surface area gets increasingly smaller in the bottom of the 1.5mL tube. Was wrong - the tube had Speedvaced to dry, probably damaging any nanoboxes in the mix at the time. Next time will Speedvac for 1hr 10min at most.

• Gel:

• • NB: "Eb" and "Fb" allude to the fact that we believe the two to have been mixed up at some point after the working stocks were made. "Eb" is believed to actually be Fb (ie. "Eb" is inward biotinylated), and vice versa.

Lane	Component	Amount
1	1kb ladder	10uL
2	p7308 (44nM)	7.5uL
3	c5.0.D wash	40uL
4	c5.0.D final	40uL
5	c5.0.8(b) wash	40uL
6	c5.0.8(b) final	40uL
7	c5.0."Eb" wash	40uL
8	c5.0."Eb" final	40uL
9	c5.0."Fb" wash	40uL
10	c5.0."Fb" final	40uL

• 

  2% agarose, EtBr, 10mM MgCl2, 1hr @ 80V, DARKER IMAGE

• 

  2% agarose, EtBr, 10mM MgCl2, 1hr @ 80V, BRIGHTER IMAGE

• Results:

• Problem: It was difficult to determine if there was a definite gel shift between p7308 and the folded structures (even just c5.0.D, which was used as a control to give a band that would run at the same rate ast the c5.0.Eb and Fb nanostructures).
  • Solution: Run the gel for another hour at 80V.

• Problem: Nothing showed up in the final-supernatants. Even if the Speedvac destroyed the nanostructures, you'd expect multimer-ed structures to be trapped near the wells or p7308-size bands and oligos. And the biotinylated oligos should definitely have shown up. This suggests several possibilities: 1) the pellets of the Speedvaced final-supernatants weren't actually dissolved; 2) the trypsinization wasn't thorough enough; 3) the trypsinization WAS thorough enough, but the F(b) and the biotinylated oligos just aren't populous enough to show up as ssDNA.
  • Solution: (2) seems to be the most likely possibility. Thus, 5uL of trypsin were added to the 50uL-reconstitution (much more concentrated, and therefore possibly more likely to produce better digestion of the streptavidin) of the streptavidin beads to try again. If the final-supernatants once again show that they were un-eluted off the beads, new trypsin might be needed - the conditions (4[[:Category:{{{1}}}|{{{1}}}]] might be not good for this formulation of the trypsin).

• Ideas for Future Work:
  • 1) Agarose Beads - higher and more efficient binding capacity
  • 2) Proteinase K - trypsin's kind of shady, not having come from the stock
  • 3) Folding at 30mM MgCl2 - might help keep boxes folded because they're folded well under the streptavidin conditions
  • 4) Using NEB Wash Buffer - instead of 1x folding buffer, NEB wash buffer might help w/ binding; DNA nanostructures evidently do pretty well in general under most conditions, so playing to the sensitivity of the streptavidin might be more appropriate

Changes for Tomorrow/Future

• Fold c6.0 - make prestock without oligo ligand and put it in working stocks.
• Quantitate p7308
• run gels:
  • with 5 μL {{etbr}
  • .5x TBE
  • 11 mM MgCl₂
  • 60V
  • 2 hours