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

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(2. Folding Rxns)
(2. Folding Rxns)
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* used two different [oligo concentrations]: 250 nM from the unconcentrated working stock, 1.5 {{uM}} from above
 
* used two different [oligo concentrations]: 250 nM from the unconcentrated working stock, 1.5 {{uM}} from above
 
* folding conditions: 80{{c}} for 2 min., decrease 1{{c}} every 2 min. for 59 more times
 
* folding conditions: 80{{c}} for 2 min., decrease 1{{c}} every 2 min. for 59 more times
 +
 +
Notes
 +
* a,b - a and b of each are the same, just two different tubes.
 +
* Oligos - 250 nM is 1x oligos, 1.5 {{um} is 6 oligos
 +
 
{| {{table}}
 
{| {{table}}
 
| align="center" style="background:#f0f0f0;"|'''Trial'''
 
| align="center" style="background:#f0f0f0;"|'''Trial'''

Revision as of 11:24, 15 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

Notes

  • a,b - a and b of each are the same, just two different tubes.
  • Oligos - 250 nM is 1x oligos, 1.5 {{um} is 6 oligos
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

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
Igemharv06 20060814 sds.jpg
lane starting amt. of nanostructures wash buffer loaded onto gel
1 7 μL 1 kb+ ladder
2 2.25 μL p7308
3 10 μL unpurified 4.0.I
4 20 μL 6hb 1x folding buffer (10 mM MgCl2) half of retentate
5 20 μL 6hb 1x folding buffer (10 mM MgCl2) w/ 0.1% SDS half of retentate
6 20 μL 4.0.I 1x folding buffer (10 mM MgCl2) half of retentate
7 20 μL 4.0.I 1x folding buffer (10 mM MgCl2) w/ 0.01% SDS half of retentate
8 20 μL 4.0.I 1x folding buffer (10 mM MgCl2) w/ 0.1% SDS half of retentate
  • ran 2% agarose gel at 80 V for 1 h. Gel appears to be of such low qualitiy that the results are inconclusive (ladder isn't clear)
  • ran another 2% agarose gel at 60V for 1 h.
  • results/discussion
    • unclear why 6hb filtration failed (oligos were retained) (lane 4)
    • 0.1% SDS gives unusual/unknown smears (lanes 5 and 8)
      • under visible light, there are thin pink-red bands in the middle of these smears. is SDS breaking down the Microcon tube plastic?
    • 0.01% SDS possibly gives higher yields than no SDS (lane 7 vs. lane 6), but it is not conclusively better yield, and it is still very poor overall yield (lane 7 vs. lane 3)

Streptavidin Bead "Protection"

  • Issues:
    • No protocol found for the Fluka agarose streptavidin beads, and the NEB magnetic bead protocol's heating requirements seem unsuitable for keeping nanoboxes folded throughout the process
      • Nanoboxes must remain folded post-elution because they must be gel-distinguishable from eluted biotinylated oligos, which we have not been able to pre-purify out by other means.
    • Elution methods are of two types:
    • 1. Overload with competitors - either streptavidin or biotinylated oligos
      • But can't: biotin-streptavidin bond is so strong that dissociation by competition would theoretically take years
    • 2. Denature the streptavidin using a) formamide, b) phenol, c) SDS + boiling
      • But can't: all the conditions require heat that might damage the nanostructures (65%degC and up)
  • Solution:
    • Degrade streptavidin with trypsin
      • Trypsin must be in solution w/o EDTA, which would otherwise chelate all the Mg2+ in the DNA nanobox solution that is necessary to keeping it folded.
  • Protocol:
1. Incubate:
     5uL beads (binding capacity: 2pmol/1uL, thus, 10pmol - far greater than the available binding sites in the DNA nanobox solutions)
     35uL 1x folding buffer
     10uL test solution

           TEST SOLUTIONS:
           --------------
           a) H2O (ie. test = no biotin in solution)
           b) biotinylated oligos (c5.0.8(b)) - 250uM per oligo in pre-working stock = 1mM biotinylation - for 1.6pmol = 1.6uL - thus, 1.6uL + 8.4uL H2O must be added for test solution
           c) c5.0 E(b) (outside biotinylation) (was Microcon "purified" Tu 8.9) - ~2000fmol of binding sites/12.5uL, or 0.16pmol/ul - thus, 1.6pmol 
           d) c5.0 F(b) (inside biotinylation) (was Microcon "purified" Tu 8.9) - "

2. Vortex
3. Pellet by drawing magnet down to bottom of tube.
4. Discard supernatant
5. Add 100uL 1x folding buffer

6. Repeat steps 2-5 three more times.

7. Trypsinize by adding to pellet:
      3uL trypsin (1mg/mL)
      27uL 1x folding buffer
8. Incubate 4hr-overnight @ 37 degrees C

9. Pellet by drawing magnet down to bottom of tube and remove supernatant to clean tube.
10. Run 20uL of each supernatant on 2% agarose gel (10mM MgCl2) for 1hr at 80V.