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.)

Prof Shih's original email said set aside some original reaction to run in the gel with the PEG-pellet and the PEG-supernatant of each of the six different conditions, and it would be nice to have two other lanes with the Microcon recovered-nanoboxes and the flowthrough. Thus one possible protocol would be the following.

(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 is done without the oligos for the lids because Prof Shih said to do this only on the c5.0 barrel.

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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