IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-10

First attempt at a protection assay

(and, in the process, more Microcon trials)

• add ligand, add latches, then Microcon, OR Microcon, add ligand, add latches
• digest
• gel analysis (PAGE)

• (Note from Tiff: Maybe it might be worth it to Microcon after the adding of ligand and latches as well, because then the excess ligand and latches then can't possibly be mistaken for the digested ligand-attachment-oligo complex.

Mg2+, Oligo-Concentration Titration, Cont.

(Note from Tiff: A remaining 20uL of each of the six reactions is in a PCR strip in one of the purple pipette-tip-box racks in the fridge, from the work on Monday. They're marked on the tube part of the strip with something like "10M 1X," all the way up to "30M 6X". However, you'll probably want to not use those and just refold two reactions each of the six reactions, so that you'll have one for PEG precipitation and one for Microcon.)

Overview

Goals

• to vary folding conditions by varying both MgCl₂ concentration and oligo concentration in order to determine which folding conditions yield the most robust and structurally sound nanostructures
• recovery yields from PEG precipitations of the products and Microcon filtration of the products will determine a) the most efficient purification protocol, as well as b) suggest which nanostructures are most robust (higher yields indicate better nanostructures)

Assumptions

• 10% PEG is a reasonable starting point for PEG precipitation trials, so we'll perform all trials with 10% PEG
• Previous results imply that the most successful Microcon trials (at least in terms of getting rid of old oligos) occur from successive spins and dilutions, since a single spin leaves many oligos. This, however, may decrease nanostructure yields. We'll use a protocol that spins and dilutes four times (the maximum the manufacture suggests) for 6 min. each (the maximum the manufacturer suggests).

Protocol overview

1. Create/choose a working stock
2. Concentrate working stock to various concentrations
3. Fold six different reactions (varying MgCl₂ concentration and oligo concentration)
4. Split each of the six reactions into two aliquots, and perform PEG precipitations on one and Microcon filtration on the other
5. Run all purification products on 2% agarose gels

Detailed protocol

1. Create/choose a working stock

Although Dr. Shih's emails indicated that we try the c5.0 barrel, we'd like to try one with a hopefully-function lid design so we can get moving on a protection assay sooner. Val and Matt chose c4.0.

2. Concentrate working stock to various concentrations

• concentrated 600 μL each of: 4.0.Fo (+latch1, +inward), 4.0.Ho (+latch2, +inward), and 4.0.Io (+latch2, +outward) in the Vacufuge until approximately 100 μL of water remained
  • each has oligo-ligand and all latches included in the working stock
  • oligo concentration is now 250 nM * 6 = 1.5 μM
  • this took several hours at 45[[:Category:{{{1}}}|{{{1}}}]]
• Io was used in the folding reactions below
  • Fo and Ho were retained for future experiments

3. Fold six different reactions (varying MgCl₂ concentration and oligo concentration)

• used three different folding buffers with varying MgCl₂ concentrations
• used two different oligo concentrations: 250 nM from the unconcentrated working stock, and 1.5 μM from the Vacufuge step above

trial	oligos	p7308 (44 nM)	folding buffer (10x)	water
1a,b	16 μL 250 nM	9 μL	4 μL 100 mM MgCl2	11 μL
2a,b	16 μL 1.5 μM	9 μL	4 μL 100 mM MgCl2	11 μL
3a,b	16 μL 250 nM	9 μL	4 μL 200 mM MgCl2	11 μL
4a,b	16 μL 1.5 μM	9 μL	4 μL 200 mM MgCl2	11 μL
5a,b	16 μL 250 nM	9 μL	4 μL 300 mM MgCl2	11 μL
6a,b	16 μL 1.5 μM	9 μL	4 μL 300 mM MgCl2	11 μL

Folding conditions: 80[[:Category:{{{1}}}|{{{1}}}]] for 2 min., decrease 1[[:Category:{{{1}}}|{{{1}}}]] every 2 min. 59 more times

4. PEG precipitations and Microcon filtrations

PEG precipitation:

• label six 1.5 mL microcentrifuge tubes 1, 2, etc.
  • use 1.5 mL tubes, and not anything smaller, so that we don't break the centrifuge
• in each one, mix (total volume is 50 μL):
  • 10 μL of the respective folded nanostructure
  • 25 μL 20% PEG (final concentration: 10%)
  • 5 μL 5 M NaCl (final concentration: 0.5 M)
  • 10 μL water
• PEG precipitation protocol:
  • incubate samples on ice for 15 min.
  • spin at 4[[:Category:{{{1}}}|{{{1}}}]] at 16k rcf for 10 min.
  • carefully pipet away supernatant
  • reconstitute pellet in 20 μL of appropriate 1x folding buffer

Microcon filtration:

• label six Microcon microcentrifuge tubes 1, 2, etc.
• pipet 10 μL folded nanostructures into six different YM-50 Microcon filters and place the filters into their respective microcentrifuge tubes
• add 490 μL water to each filter
• spin at 14k rcf for 6 min.
  • this should leave ~50 μL remaining (according to yesterday's experiments)
• add 440 μL water, spin, add water, spin, add water, spin (total of four spins)
• retentate recovery: invert filter into a clean, labeled Microcon microcentrifuge tube and spin at 1k rcf for 3 min.

5. Gel analysis

Depending on Microcon retentate volumes, it may be necessary to normalize the volumes run in the gel. If that's the case, then run half of all the volumes listed.

To save time, space, and sanity, we won't run the supernatants and flow-throughs, since they don't give us much information about yield and purity, and since they'll require lots of Vacufuging in order to obtain normalized samples that will fit in a gel.

On a 2% agarose gel run in TBE, with both the gel and buffer supplemented to 10 mM MgCl₂, at 60V for 1 h:

2% agarose gel run in TBE, with both the gel and buffer supplemented to 10 mM MgCl₂, at 60V for 1 h

Lane	Trial	Purification	Amount
1	30 nM oligos, 10 mM MgCl2	unpurified	10 μL
2	30 nM oligos, 10 mM MgCl2	PEG pellet	all (20 μL)
3	30 nM oligos, 10 mM MgCl2	Microcon retentate	all
4	30 nM oligos, 20 mM MgCl2	unpurified	10 μL
5	30 nM oligos, 20 mM MgCl2	PEG pellet	all (20 μL)
6	30 nM oligos, 20 mM MgCl2	Microcon retentate	all
7	30 nM oligos, 30 mM MgCl2	unpurified	10 μL
8	30 nM oligos, 30 mM MgCl2	PEG pellet	all (20 μL)
9	30 nM oligos, 30 mM MgCl2	Microcon retentate	all
10	1 Kb+ ladder	n/a	10 μL
11	p7308 (44 nM)	n/a	4.5 μL
12	600 nM oligos, 10 mM MgCl2	unpurified	10 μL
13	600 nM oligos, 10 mM MgCl2	PEG pellet	all (20 μL)
14	600 nM oligos, 10 mM MgCl2	Microcon retentate	all
15	600 nM oligos, 20 mM MgCl2	unpurified	10 μL
16	600 nM oligos, 20 mM MgCl2	PEG pellet	all (20 μL)
17	600 nM oligos, 20 mM MgCl2	Microcon retentate	all
18	600 nM oligos, 30 mM MgCl2	unpurified	10 μL
19	600 nM oligos, 30 mM MgCl2	PEG pellet	all (20 μL)
20	600 nM oligos, 30 mM MgCl2	Microcon retentate	all

Results/discussion

• both PEG precipitation and Microcon filtration appear to effectively remove free oligos
• smearing of nanostructure bands indicates that PEG precipitation appears to damage nanostructures independently of oligo and MgCl₂ concentration
• Microcon filtration continues to afford low nanostructure yields

Earlier protocol guidelines

(1) Concentrate enough 6x oligos for 6 samples.

• In other words, make up 3 tubes of c5.0A without lids.
  • 36 reactions-worth of oligos are needed.
  • Each tube of working stock that we make has enough for 12.5 reactions (200uL/16uL of oligos per folding reaction = 12.5)
  • This should be done without the oligos for the lids because Prof Shih said to do this only on the c5.0 barrel. (However, I might be misinterpreting the descriptions of the pre-working stocks, so please check the following table before you go ahead and mix up the working stocks.)

Stock ID	Experiment	1	2	3	4	4L	5	5L	6	7	8	9	10+11	12	13+14	15	16+17	18	19	20	21	Total	H2O Needed
c5.0A	no latches, no aptamers	172uL	-	-	5uL	-	5uL	-	3uL	3uL	-	-	-	-	-	-	-	-	-	-	-	188uL	12uL

• Concentrate 6 tubes of 6-reactions-worth-of-oligos in each tube.
  • Pipette 96uL (6 x 16uL = 96uL) of the working stock you made up into each of 6 tubes.
  • Speedvac for approximately 45 minutes, or until dry. (Don't worry, drying oligos isn't bad - just nanoboxes.)
  • Reconstitute in 16uL of dH2O.

(2) Fold two of each of the 6 reactions.

• • Tubes labeled "10x folding buffer" (which is the normal kind, with 100mM of MgCl2 in it), "10x folding buffer 200mM MgCl2, and "10x folding buffer 300mM MgCl2" should be in the fridge somewhere. The 200mM and 300mM should be in the same rack.
    • Into each of the six tubes you've just Speedvaced, add:
      • 9uL of p7308 (44nM)
      • 4uL of the appropriate 10x folding buffer (see table).
      • 11uL of H2O
    • Pipette each of these 40uL of reaction into PCR tubes.

• • Into six other PCR tubes, mix:
    • • 9uL of p7308 (44nM)
      • 4uL of the appropriate 10x folding buffer (see table).
      • 11uL of H2O
      • 16ul of c5.0 A, without lids. The two tubes in the c5.0 rack labelled "c5.0 A (no lids)" should be fine.

• • Fold using FOLDINGD program in thermocycler.

(3) Purify using (A)PEG precipitation, and (B)Microcon tubes.

• (A) For PEG purification:
  • Use 30uL from each of the six types of reactions.

In a 1.5mL tube (so that you'll be able to spin in a centrifuge without damage later), add:
   20uL 20% PEG
   10uL 2.5M NaCl
Ice for 15 min
Spin at 4 deg C for 10 min
Remove supernatant (50uL) to a separate tube
Reconstitute pellet in 30uL H2O

• (B) For Microcon purification:
  • Use 30uL from each of the six types of reactions.
  • Use the Microcon protocol that is yielding the best results - 4 washes, spin for 6min each time seems to be the best so far.
  • After the recovery, assess the amount of liquid you've recovered; if below 30uL, add H2O till you reach 30uL. This will allow equal comparisons in the gel later.

(4) Run two 20-lane 2% 10mM MgCl2 agarose gels (everything won't fit on just one).

• Only 10uL of each of the 30uL PEG-pellet or 30uL of Microcon "elute" will be used in the gel, so that 20uL will be left for EM or whatever other final use they might be useful for.
• 10uL of each of the original reactions will be used in the gels.
  • This is to try to keep equivalent nanobox-concentrations with the other 10uL used for the PEG-pellet and Microcon "elute" lanes.
  • Because the pellet and elute were reconsistuted to the original 30uL taken from the reactions before they were purified, 10uL of the original = 10uL of the purified form, theoretically.

• The following two gels should be run at 60V for 1hr:

lane	component	[component]	amount
0	1kb ladder	1x	10uL
1	p7308	44nM	7.5uL
2	10mM Mg, 1x unpurified	99fM	10uL
3	10mM Mg, 1x PEG-pellet	~99fM	10uL
4	10mM Mg, 1x PEG-supernatant	-	~40uL
5	10mM Mg, 1x Microcon "elute"	~99fM	10uL
6	10mM Mg, 1x Microcon flowthrough	-	~40uL
7	20mM Mg, 1x unpurified	99fM	10uL
8	20mM Mg, 1x PEG-pellet	~99fM	10uL
9	20mM Mg, 1x PEG-supernatant	-	~40uL
10	20mM Mg, 1x Microcon "elute"	~99fM	10uL
11	20mM Mg, 1x Microcon flowthrough	-	~40uL
12	30mM Mg, 1x unpurified	99fM	10uL
13	30mM Mg, 1x PEG-pellet	~99fM	10uL
14	30mM Mg, 1x PEG-supernatant	-	~40uL
15	30mM Mg, 1x Microcon "elute"	~99fM	10uL
16	30mM Mg, 1x Microcon flowthrough	-	~40uL
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And,

lane	component	[component]	amount
0	1kb ladder	1x	10uL
1	p7308	44nM	7.5uL
2	10mM Mg, 6x unpurified	99fM	10uL
3	10mM Mg, 6x PEG-pellet	~99fM	10uL
4	10mM Mg, 6x PEG-supernatant	-	~40uL
5	10mM Mg, 6x Microcon \"elute\"	~99fM	10uL
6	10mM Mg, 6x Microcon flowthrough	-	~40uL
7	20mM Mg, 6x unpurified	99fM	10uL
8	20mM Mg, 6x PEG-pellet	~99fM	10uL
9	20mM Mg, 6x PEG-supernatant	-	~40uL
10	20mM Mg, 6x Microcon \"elute\"	~99fM	10uL
11	20mM Mg, 6x Microcon flowthrough	-	~40uL
12	30mM Mg, 6x unpurified	99fM	10uL
13	30mM Mg, 6x PEG-pellet	~99fM	10uL
14	30mM Mg, 6x PEG-supernatant	-	~40uL
15	30mM Mg, 6x Microcon \"elute\"	~99fM	10uL
16	30mM Mg, 6x Microcon flowthrough	-	~40uL
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Thrombin bead assay

Folded design 3.2.E (aptamers outside, no latches) for use in a future assay in which we test whether the nanostructures are capable of binding thrombin beads.