IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-22

Design 5
Realized that lid oligos were accidentally added in folding of c5.0.A on 8-20. Refolding c5.0.A to be just barrel oligos. Will use in PEG fractionation.

Protocol
Testing Conditions: Test Solutions: Mix: 28uL agarose streptavidin beads 40uL of each test solution rxn
 * (A) Unbeaded
 * (B) Beaded
 * (C) Free-streptavidin incubated
 * (D) Free-streptavidin incubated, then beaded
 * (1) c5.0.A (lidless barrel, no biotinylation)
 * (2) c5.0.Eb (lidless, outside biotinylation)
 * (3) c5.0.Fb (lidless, inside biotinylation)
 * (B) Beaded

Spin down; remove supernatant to new tube. Speedvac to ~40uL Load 40uL in gel

2uL of 2uM streptavidin 40uL of test solution 26uL of H2O
 * (C) Free-streptavidin incubated
 * Free-streptavidin concentration needed
 * 1680pmoles on biotinylated boxes + 36pmoles of free biotinylated oligos = 1716pmoles = 1.7nmoles
 * free streptavidin @ 2uM = 2nmol/uL
 * thus, for safety, include 2uL of streptavidin

Speedvac to ~40uL Load 40uL in gel

2uL of 2uM streptavidin 40uL of test solution
 * (D) Free-streptavidin incubated, then beaded

Wait 15 minutes

Add 28uL of agarose streptavidin beads Wait 5 minutes

Spin down; remove supernatant to new tube. Speedvac to ~40uL Load 40uL in gel

Gel

 * Gel was 2% agarose in 0.5xTBE, 5uL EtBr, 10mM MgCl2. Ran for 1.5hr @ 80V in 0.5xTBE supplemented to 10mM MgCl2.


 * Preliminary Conclusions:
 * no difference seen after adding free-streptavidin, and then beading to the outside biotinylated box (lane 13)
 * possible not enough free-streptavidin used
 * also, no difference in gel band brightness from untreated to beaded - agarose beads either do not work as well as expected (maybe should use magnetic beads), or the amount used was too low