IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-14

Goals for today

Microcon Purification Tweaking

• repeat Friday's mega PEG ppt on 5.0 (?)
• Micron experiments with 0.1% and 0.01% SDS in buffer
  • ...and use 1x folding buffer and not water for washes
  • also: perform control expt with 10 bp+ ladder, since according to Millipore documentation, the filter should retain ds DNAs longer than 100 bp

Streptavidin-Bead "Protection" Assay on Inside- and Outside-Biotinylated c5.0

• NB: no good purification of nanostructure from oligo has been achieved, but gel separation after elution should differentiate formerly bead-bound oligos from formerly bead-bound nanostructures

Redux of [Mg++], [oligos]

• Based on the inconclusive gels from last Monday, the titration will be redone with c5.0 at 8% PEG, 0.5M NaCl final.

Mg2+, Oligo-Concentration Titration w/ c5.0

Goals

• vary folding conditions ([MgCl₂] and [oligo]) in order to determine best folding conditions for c5.0
• determine most efficient purification protocol (Microcon vs. PEG) based on recovery yields

Protocol

1. Working Stock Concentration

• concentrated 6 tubes of 96 μL c5.0D.L (no latches, outside-bound ligand) in Vacufuge so that [oligo]= 250nM * 6 = 1.5 μM

2. Folding Rxns

• used three different folding buffers varying [MgCl₂]
• used two different [oligo concentrations]: 250 nM from the unconcentrated working stock, 1.5 μM from above
• folding conditions: 80[[:Category:{{{1}}}|{{{1}}}]] for 2 min., decrease 1[[:Category:{{{1}}}|{{{1}}}]] every 2 min. for 59 more times

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

3. PEG & Microcon

PEG:

• GOAL: test 8% and 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 20 μL of water

Microcon w/ detergent

• add 20 μL given nanostructure to center of YM-50 Micrcon tube
• add 480 μL given folding buffer, microcentrifuge for 6 min. at 14k rcf, and repeat dilution and spinning 4 more times
• yielded approx. 100 μL retentate, which was concentrated to 15 to 60 μL in a Vacufuge (about 30 min. at 45 [[:Category:{{{1}}}|{{{1}}}]]), depending on the sample

lane	starting amt. of nanostructures	wash buffer	loaded onto gel
1			5 μL 1 kb+ ladder
2			2.25 μL p7308 and 10 μL unpurified 4.0.I
3	20 μL 6hb	1x folding buffer (10 mM MgCl2)	half of retentate
4	20 μL 6hb	1x folding buffer (10 mM MgCl2) w/ 0.1% SDS	half of retentate
5	20 μL 4.0.I	1x folding buffer (10 mM MgCl2)	half of retentate
6	20 μL 4.0.I	1x folding buffer (10 mM MgCl2) w/ 0.01% SDS	half of retentate
7	20 μL 4.0.I	1x folding buffer (10 mM MgCl2) w/ 0.1% SDS	half of retentate