IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-22: Difference between revisions
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==Streptavidin Depletion Assay== | ==Streptavidin Depletion Assay== | ||
===Protocol=== | ===Protocol=== | ||
'''Testing Conditions:''' | |||
***''(A) Unbeaded'' | ***''(A) Unbeaded'' | ||
***''(B) Beaded'' | ***''(B) Beaded'' | ||
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***(2) c5.0.Eb (lidless, outside biotinylation) | ***(2) c5.0.Eb (lidless, outside biotinylation) | ||
***(3) c5.0.Fb (lidless, inside biotinylation) | ***(3) c5.0.Fb (lidless, inside biotinylation) | ||
'''Mix:''' | |||
*''(B) Beaded'' | |||
<pre> | <pre> | ||
28uL agarose streptavidin beads | 28uL agarose streptavidin beads | ||
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</pre> | </pre> | ||
*''(C) Free-streptavidin incubated'' | |||
****Free-streptavidin concentration needed | ****Free-streptavidin concentration needed | ||
*****1680pmoles on biotinylated boxes + 36pmoles of free biotinylated oligos = 1716pmoles = 1.7nmoles | *****1680pmoles on biotinylated boxes + 36pmoles of free biotinylated oligos = 1716pmoles = 1.7nmoles | ||
| Line 50: | Line 50: | ||
</pre> | </pre> | ||
*''(D) Free-streptavidin incubated, then beaded'' | |||
<pre> | <pre> | ||
2uL of 2uM streptavidin | 2uL of 2uM streptavidin | ||
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Load 40uL in gel | Load 40uL in gel | ||
</pre> | </pre> | ||
===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. | * Gel was 2% agarose in 0.5xTBE, 5uL EtBr, 10mM MgCl2. Ran for 1.5hr @ 80V in 0.5xTBE supplemented to 10mM MgCl2. | ||
Revision as of 14:44, 22 August 2006
Folding
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.
| Working Stocks | Description | Pre-Working Stocks | Water | Total |
| c5.0A | no latches, no aptamers, no lids | 1 (86 μL), 4 (2.5 μL), 5 (2.5 μL), 6 (1.5 μL), 7 (1.5 μL) | 106 μL | 200 μL |
Streptavidin Depletion Assay
Protocol
Testing Conditions:
- (A) Unbeaded
- (B) Beaded
- (C) Free-streptavidin incubated
- (D) Free-streptavidin incubated, then beaded
- Test Solutions:
- (1) c5.0.A (lidless barrel, no biotinylation)
- (2) c5.0.Eb (lidless, outside biotinylation)
- (3) c5.0.Fb (lidless, inside biotinylation)
Mix:
- (B) Beaded
28uL agarose streptavidin beads 40uL of each test solution rxn Spin down; remove supernatant to new tube. Speedvac to ~40uL Load 40uL in gel
- (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
- Free-streptavidin concentration needed
2uL of 2uM streptavidin 40uL of test solution 26uL of H2O Speedvac to ~40uL Load 40uL in gel
- (D) Free-streptavidin incubated, then beaded
2uL of 2uM streptavidin 40uL of test solution 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.
