IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-25
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PEG fractionation
- Goal: to get cleaner purification of oligos away from nanostructures and to increase the volume of purified nanostructures we have for protection assays. Given the results of 8-23 I'm working with a new batch of folded design 5 and trying to see if the double bands and trouble moving through the gel were simply artifacts of that particular folding or if they are larger problems that need to be addressed.
- Nanostructures:
- using 30mM MgCl2, 1x oligos (seems to work best given previous results).
- designs: c5.0.A, c5.0.C, c5.0.D
- PEG: 8%, 10%. Total volume in each is 100 μL
- 8 % Cocktail:
40 μL 20% PEG
10 μL 5M NaCl
10 μL water
40 μL Nanostructures (add last)
- 10 % Cocktail:
50 μL 20% PEG
10 μL 5M NaCl
40 μL Nanostructures (add last)
- incubate on ice for 15 min.
- spin at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
- carefully pipette off supernatant
- resuspended "pellet" in 1x folding buffer. for now, resuspend in original total volume (100 μL but may resuspend in less in the future to improve protection assay results)
- note: add PEG first, nanostructures last; mix using tapping after everything added. let sit for ~5 min. before putting it on ice
| Lane | contents |
| gel1-1 | ladder |
| gel1-2 | scaffold |
| gel1-3 | 5.0.A unpurified |
| gel1-4 | 5.0.A 8% pellet 1 |
| gel1-5 | 5.0.A 8% supernatant 1 |
| gel1-6 | 5.0.A 8% pellet 2 |
| gel1-7 | 5.0.A 8% supernatant 2 |
| gel1-8 | 5.0.A 10% pellet 1 |
| gel1-9 | 5.0.A 10% supernatant 1 |
| gel1-10 | 5.0.A 10% pellet 2 |
| gel1-11 | 5.0.A 10% supernatant 2 |
| gel1-12 | 5.0.C unpurified |
| gel1-13 | 5.0.C 8% pellet 1 |
| gel1-14 | 5.0.C 8% supernatant 1 |
| gel1-15 | 5.0.C 8% pellet 2 |
| gel1-16 | 5.0.C 8% supernatant 2 |
| gel1-17 | 5.0.C 10% pellet 1 |
| gel1-18 | 5.0.C 10% supernatant 1 |
| gel1-19 | 5.0.C 10% pellet 2 |
| gel1-20 | 5.0.C 10% supernatant 2 |
| gel2-1 | ladder |
| gel2-2 | 5.0.D unpurified |
| gel2-3 | 5.0.D 8% pellet 1 |
| gel2-4 | 5.0.D 8% supernatant 1 |
| gel2-5 | 5.0.D 8% pellet 2 |
| gel2-6 | 5.0.D 8% supernatant 2 |
| gel2-7 | 5.0.D 10% pellet 1 |
| gel2-8 | 5.0.D 10% supernatant 1 |
| gel2-9 | 5.0.D 10% pellet 2 |
| gel2-10 | 5.0.D 10% supernatant 2 |
Strep Treatment Protocol
- Same as that done yesterday, only with just c5.0.A and c5.0.Fb folded yesterday.
Gel
- NB: Accidentally ran old-folded Fb before new-folded Fb in the untreated trial. New-folded Fb was loaded into the gel before old-folded Fb in every other trial.
| Lane | Component | Test Condition | Amount |
| 1 | 1kb+ ladder | - | 10uL |
| 2 | p7308 (~42nM) | - | 9uL |
| 3 | c5.0.A barrel (lidless) | Untreated | 10uL |
| 4 | inside biotinylated barrel (folded 8.22) | Untreated | 10uL |
| 5 | inside biotinylated barrel (folded 8.24) | Untreated | 10uL |
| 6 | barrel (lidless) | Beaded | ~38uL |
| 7 | inside biotinylated barrel (folded 8.24) | Beaded | ~38uL |
| 8 | inside biotinylated barrel (folded 8.22) | Beaded | ~38uL |
| 9 | barrel (lidless) | Free-strep | ~38uL |
| 10 | inside biotinylated barrel (folded 8.24) | Free-strep | ~38uL |
| 11 | inside biotinylated barrel (folded 8.22) | Free-strep | ~38uL |
| 12 | barrel (lidless) | Free, bead | ~38uL |
| 13 | inside biotinylated barrel (folded 8.24) | Free, bead | ~38uL |
| 14 | inside biotinylated barrel (folded 8.22) | Free, bead | ~38uL |
