Difference between revisions of "IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-14"

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(Microcon w/ detergent)
(Microcon w/ detergent)
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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||||||7 {{ul}} 1 kb+ ladder
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| 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 12:00, 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]



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