IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-14: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''loaded onto gel''' | | align="center" style="background:#f0f0f0;"|'''loaded onto gel''' | ||
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| 1|||||| | | 1||||||5 {{ul}} 1 kb+ ladder | ||
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| 2||||||2.25 {{ul}} p7308 and 10 {{ul}} unpurified 4.0.I | | 2||||||2.25 {{ul}} p7308 and 10 {{ul}} unpurified 4.0.I |
Revision as of 13:00, 14 August 2006
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 |