IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-15

Mg2+, Oligo-Concentration Titration w/ c5.0 (cont'd)

3. PEG

PEG:

• GOAL: test 6%, 8%, 10% PEG precipitations
• incubated on ice for 15 min.
• spun at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
• carefully pipetted off supernatant
• resuspended "pellet" in 50 μL of water

• 

  Gel 1 after 45 minutes

• 

  Gel 2 after 45 minutes

• 

  Gel 1 after 75 minutes

• 

  Gel 2 after 75 minutes

RESULTS:

• decent separation of pellet and oligos for most of the trials
• looking at gels after 75 min. seems to show that the nanostructures are going into the gel, so we don't think they're getting denatured
• best trials seem to be: Trial 4 (20 mM MgCl₂ w/ 6x oligos) and Trial 5 (30 mM MgCl₂ w/ 1x oligos), each at 8% and 10% PEG
• next step: we will repeat this experiment in larger quantities to prepare for a protection assay

Trial	Final PEG %	Lanes	20% PEG (μL)	5 M NaCl (μL)	Nanostructures (μL)	Water (μL)	Total volume (μL)
1kb+ ladder	-	1	-	-	-	-	10
p7308	-	2	-	-	-	-	10
1	0%	3 (untreated trial 1)	-	-	-	-	-
1-6	6%	4 (pellet)	15	5	20	10	50
1-6	6%	5 (supernatant)	-	-	-	-	-
1-8	8%	6 (pellet)	20	5	20	5	50
1-8	8%	7 (supernatant)	-	-	-	-	-
1-10	10%	8 (pellet)	25	5	20	0	50
1-10	10%	9 (supernatant)	-	-	-	-	-
2	0%	10 (untreated trial 2)	-	-	-	-	-
2-6	6%	11 (pellet)	15	5	20	10	50
2-6	6%	12 (supernatant)	-	-	-	-	-
2-8	8%	13 (pellet)	20	5	20	5	50
2-8	8%	14 (supernatant)	-	-	-	-	-
2-10	10%	15 (pellet)	25	5	20	0	50
2-10	10%	16 (supernatant)	-	-	-	-	-
3	0%	17 (untreated trial 3)	-	-	-	-	-
3-6	6%	18 (pellet)	15	5	20	10	50
3-6	6%	19 (supernatant)	-	-	-	-	-
1kb+ ladder	-	1	-	-	-	-	10
p7308	-	2	-	-	-	-	10
3-8	8%	3 (pellet)	20	5	20	5	50
3-8	8%	4 (supernatant)	-	-	-	-	-
3-10	10%	5 (pellet)	25	5	20	0	50
3-10	10%	6 (supernatant)	-	-	-	-	-
4	0%	7 (untreated trial 4)	-	-	-	-	-
4-6	6%	8 (pellet)	15	5	20	10	50
4-6	6%	9 (supernatant)	-	-	-	-	-
4-8	8%	10 (pellet)	20	5	20	5	50
4-8	8%	11 (supernatant)	-	-	-	-	-
4-10	10%	12 (pellet)	25	5	20	0	50
4-10	10%	13 (supernatant)	-	-	-	-	-
5	0%	14 (untreated trial 5)	-	-	-	-	-
5-6	6%	15 (pellet)	15	5	20	10	50
5-6	6%	16 (supernatant)	-	-	-	-	-
5-8	8%	17 (pellet)	20	5	20	5	50
5-8	8%	18 (supernatant)	-	-	-	-	-
5-10	10%	19 (pellet)	25	5	20	0	50
5-10	10%	20 (supernatant)	-	-	-	-	-
1kb+ ladder	-	1	-	-	-	-	10
p7308	-	2	-	-	-	-	10
6	0%	3 (untreated trial 6)	-	-	-	-
6-6	6%	4 (pellet)	15	5	20	10	50
6-6	6%	5 (supernatant)	-	-	-	-	-
6-8	8%	6 (pellet)	20	5	20	5	50
6-8	8%	7 (supernatant)	-	-	-	-	-
6-10	10%	8 (pellet)	25	5	20	0	50
6-10	10%	9 (supernatant)	-	-	-	-	-
6hb-4	4%	10 (pellet)	10	5	20	15	50
6hb-4	4%	11 (supernatant)	-	-	-	-	-
6hb-6	6%	12 (pellet)	15	5	20	10	50
6hb-6	6%	13 (supernatant)	-	-	-	-	-
6hb-8	8%	14 (pellet)	20	5	20	5	50
6hb-8	8%	15 (supernatant)	-	-	-	-	-
6hb-10	10%	16 (pellet)	25	5	20	0	50
6hb-10	10%	17 (supernatant)	-	-	-	-	-

Revised protection assay protocol (proposed)

Folding

• use working stock that includes all latches, as well as oligo-ligand
• fold nanostructures with appropriate folding conditions
  • appears to be either: 30 mM MgCl₂ with 1x oligos or 20 mM MgCl₂ with 6x, depending on PEG fractionation repeat experiment

Purification

• purify nanostructures with PEG precipitation using 7%, 8%, 9%, or 10%, depending on PEG fractionation repeat experiment

Digest

• total volume of each digest trial: 20 μL

trial	DNA	10x NEBuffer 4	10x BSA	AscI	water
experimental	10 μL 10 nM purified, ligand-incubated nanostructures	2 μL	2 μL	1 μL 500 U/mL	5 μL
-oligos	2.25 μL 44 nM p7308 (or appropriate scaffold)	2 μL	2 μL	1 μL 500 U/mL	13.75 μL
-enzyme	10 μL 10 nM purified, ligand-incubated nanostructures	2 μL	2 μL	0 μL	6 μL
-ligand	10 μL 10 nM purified nanostructures (no ligand)	2 μL	2 μL	1 μL 500 U/mL	5 μL
-nanostructures -enzyme	1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand	2 μL	2 μL	0 μL	14 μL
-nanostructures	1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand	2 μL	2 μL	1 μL 500 U/mL	13 μL

p7308 quantitation

• Speedvac 060522 p7308 sample down to 50% volume. This should remove any ethanol, and give you a slightly more manageable volume.
• Pour 2% agraose, 11 mM MgCl₂ gel
• For gel loading make 1:2 dilution (add 20 μL of p7308 to 20 μL dH₂O). Original estimate for 060522 prep was 42 nM, so hopefully it should correspond pretty well to 44 nM sample.
• Load gel according to table below
• Run for 2 hrs, 70V
• When imaging gel, use spot density tool to measure intensity of each band
  • Use saturation indicator to take a picture just below the point where any bands start saturating on the image
  • Draw a rectangle that fits around the largest band on the gel
  • Copy that rectangle and position it directly above the first band. This will be used to measure background
  • Repeat this for every band on the gel (one box for the band, one box for background)
  • Record this data along with gel picture on the wiki
• To determine p7308 concentration, use background-subtracted value for each volume. Scale each unknown concentration against the control (44 nM) according to the ratio of background-subtracted intensity for the band that looks closest in intensity
  • For example, if the band in lane 1 (3 μL, 44 nM) had an intensity of 1000, and the band in lane 5 (3 μL, ?? nM) had an intensity of 900, then we would record 900/1000 * 44 = 39.6 nM as the estimated concentration for that lane. Repeating for each lane should give you 3 data points, which you can average (throwing out any obvious outliers).

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

Lane	Contents	Loading Buffer
0	1kb DNA ladder (5 μL)
1	p7308 060323, 44 nM (3 μL)	AGLB (2 μL) + dH2O (9 μL)
2	p7308 060323, 44 nM (6 μL)	AGLB (2 μL) + dH2O (6 μL)
3	p7308 060323, 44 nM (9 μL)	AGLB (2 μL) + dH2O (3 μL)
4	p7308 060522, 1:2 dil (1 μL)	AGLB (2 μL) + dH2O (11 μL)
5	p7308 060522, 1:2 dil (3 μL)	AGLB (2 μL) + dH2O (9 μL)
6	p7308 060522, 1:2 dil (6 μL)	AGLB (2 μL) + dH2O (6 μL)
7	p7308 060522, 1:2 dil (9 μL)	AGLB (2 μL) + dH2O (3 μL)
8	p7308 060522, 1:2 dil (12 μL)	AGLB (2 μL)