IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-15
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Mg2+, Oligo-Concentration Titration w/ c5.0 (cont'd)
3. PEG
PEG:
- GOAL: test 6%, 8%, 10% PEG precipitations on c5.0.D - also evaluating differences between the concentrations of MgCl2
- Trials:
Trial 1 - 10mM MgCl2, 1x oligos
Trial 2 - 10 mM MgCl2, 6x oligos
Trial 3 - 20 mM MgCl2, 1x oligos
Trial 4 - 20 mM MgCl2, 6x oligos
Trial 5 - 30 mM MgCl2, 1x oligos
Trial 6 - 30 mM MgCl2, 6x oligos
- 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 50 μL of water
- note: we added PEG first, nanostructures last; mixed using tapping after everything was added; let it sit for like...5 min. before putting it on ice; let it sit a bit (ie. 10 min.) after centrifuging
-
Gel 1 after 45 minutes
-
Gel 2 after 45 minutes
-
Gel 1 after 75 minutes
-
Gel 2 after 75 minutes
RESULTS:
- decent separation of pellet and oligos for most of the trials
- looking at gels after 75 min. seems to show that the nanostructures are going into the gel, so we don't think they're getting denatured
- best trials seem to be: Trial 4 (20 mM MgCl2 w/ 6x oligos) and Trial 5 (30 mM MgCl2 w/ 1x oligos), each at 8% and 10% PEG
- next step: we will repeat this experiment in larger quantities to prepare for a protection assay
| Trial | Final PEG % | Lanes | 20% PEG (μL) | 5 M NaCl (μL) | Nanostructures (μL) | Water (μL) | Total volume (μL) |
| 1kb+ ladder | - | 1 | - | - | - | - | 10 |
| p7308 | - | 2 | - | - | - | - | 10 |
| 1 | 0% | 3 (untreated trial 1) | - | - | - | - | - |
| 1-6 | 6% | 4 (pellet) | 15 | 5 | 20 | 10 | 50 |
| 1-6 | 6% | 5 (supernatant) | - | - | - | - | - |
| 1-8 | 8% | 6 (pellet) | 20 | 5 | 20 | 5 | 50 |
| 1-8 | 8% | 7 (supernatant) | - | - | - | - | - |
| 1-10 | 10% | 8 (pellet) | 25 | 5 | 20 | 0 | 50 |
| 1-10 | 10% | 9 (supernatant) | - | - | - | - | - |
| 2 | 0% | 10 (untreated trial 2) | - | - | - | - | - |
| 2-6 | 6% | 11 (pellet) | 15 | 5 | 20 | 10 | 50 |
| 2-6 | 6% | 12 (supernatant) | - | - | - | - | - |
| 2-8 | 8% | 13 (pellet) | 20 | 5 | 20 | 5 | 50 |
| 2-8 | 8% | 14 (supernatant) | - | - | - | - | - |
| 2-10 | 10% | 15 (pellet) | 25 | 5 | 20 | 0 | 50 |
| 2-10 | 10% | 16 (supernatant) | - | - | - | - | - |
| 3 | 0% | 17 (untreated trial 3) | - | - | - | - | - |
| 3-6 | 6% | 18 (pellet) | 15 | 5 | 20 | 10 | 50 |
| 3-6 | 6% | 19 (supernatant) | - | - | - | - | - |
| 1kb+ ladder | - | 1 | - | - | - | - | 10 |
| p7308 | - | 2 | - | - | - | - | 10 |
| 3-8 | 8% | 3 (pellet) | 20 | 5 | 20 | 5 | 50 |
| 3-8 | 8% | 4 (supernatant) | - | - | - | - | - |
| 3-10 | 10% | 5 (pellet) | 25 | 5 | 20 | 0 | 50 |
| 3-10 | 10% | 6 (supernatant) | - | - | - | - | - |
| 4 | 0% | 7 (untreated trial 4) | - | - | - | - | - |
| 4-6 | 6% | 8 (pellet) | 15 | 5 | 20 | 10 | 50 |
| 4-6 | 6% | 9 (supernatant) | - | - | - | - | - |
| 4-8 | 8% | 10 (pellet) | 20 | 5 | 20 | 5 | 50 |
| 4-8 | 8% | 11 (supernatant) | - | - | - | - | - |
| 4-10 | 10% | 12 (pellet) | 25 | 5 | 20 | 0 | 50 |
| 4-10 | 10% | 13 (supernatant) | - | - | - | - | - |
| 5 | 0% | 14 (untreated trial 5) | - | - | - | - | - |
| 5-6 | 6% | 15 (pellet) | 15 | 5 | 20 | 10 | 50 |
| 5-6 | 6% | 16 (supernatant) | - | - | - | - | - |
| 5-8 | 8% | 17 (pellet) | 20 | 5 | 20 | 5 | 50 |
| 5-8 | 8% | 18 (supernatant) | - | - | - | - | - |
| 5-10 | 10% | 19 (pellet) | 25 | 5 | 20 | 0 | 50 |
| 5-10 | 10% | 20 (supernatant) | - | - | - | - | - |
| 1kb+ ladder | - | 1 | - | - | - | - | 10 |
| p7308 | - | 2 | - | - | - | - | 10 |
| 6 | 0% | 3 (untreated trial 6) | - | - | - | - | |
| 6-6 | 6% | 4 (pellet) | 15 | 5 | 20 | 10 | 50 |
| 6-6 | 6% | 5 (supernatant) | - | - | - | - | - |
| 6-8 | 8% | 6 (pellet) | 20 | 5 | 20 | 5 | 50 |
| 6-8 | 8% | 7 (supernatant) | - | - | - | - | - |
| 6-10 | 10% | 8 (pellet) | 25 | 5 | 20 | 0 | 50 |
| 6-10 | 10% | 9 (supernatant) | - | - | - | - | - |
| 6hb-4 | 4% | 10 (pellet) | 10 | 5 | 20 | 15 | 50 |
| 6hb-4 | 4% | 11 (supernatant) | - | - | - | - | - |
| 6hb-6 | 6% | 12 (pellet) | 15 | 5 | 20 | 10 | 50 |
| 6hb-6 | 6% | 13 (supernatant) | - | - | - | - | - |
| 6hb-8 | 8% | 14 (pellet) | 20 | 5 | 20 | 5 | 50 |
| 6hb-8 | 8% | 15 (supernatant) | - | - | - | - | - |
| 6hb-10 | 10% | 16 (pellet) | 25 | 5 | 20 | 0 | 50 |
| 6hb-10 | 10% | 17 (supernatant) | - | - | - | - | - |
Revised protection assay protocol (proposed)
Folding
- use working stock that includes all latches, as well as oligo-ligand
- fold nanostructures with appropriate folding conditions
- appears to be either: 30 mM MgCl2 with 1x oligos or 20 mM MgCl2 with 6x, depending on PEG fractionation repeat experiment
Purification
- purify nanostructures with PEG precipitation using 7%, 8%, 9%, or 10%, depending on PEG fractionation repeat experiment
Digest
- total volume of each digest trial: 20 μL
| trial | DNA | 10x NEBuffer 4 | 10x BSA | AscI | water |
| experimental | 10 μL 10 nM purified, ligand-incubated nanostructures | 2 μL | 2 μL | 1 μL 500 U/mL | 5 μL |
| -oligos | 2.25 μL 44 nM p7308 (or appropriate scaffold) | 2 μL | 2 μL | 1 μL 500 U/mL | 13.75 μL |
| -enzyme | 10 μL 10 nM purified, ligand-incubated nanostructures | 2 μL | 2 μL | 0 μL | 6 μL |
| -ligand | 10 μL 10 nM purified nanostructures (no ligand) | 2 μL | 2 μL | 1 μL 500 U/mL | 5 μL |
| -nanostructures -enzyme | 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand | 2 μL | 2 μL | 0 μL | 14 μL |
| -nanostructures | 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand | 2 μL | 2 μL | 1 μL 500 U/mL | 13 μL |
p7308 quantitation
- Speedvac 060522 p7308 sample down to 50% volume. This should remove any ethanol, and give you a slightly more manageable volume.
- Pour 2% agraose, 11 mM MgCl2 gel
- For gel loading make 1:2 dilution (add 20 μL of p7308 to 20 μL dH2O). Original estimate for 060522 prep was 42 nM, so hopefully it should correspond pretty well to 44 nM sample.
- Load gel according to table below
- Run for 2 hrs, 70V
- When imaging gel, use spot density tool to measure intensity of each band
- Use saturation indicator to take a picture just below the point where any bands start saturating on the image
- Draw a rectangle that fits around the largest band on the gel
- Copy that rectangle and position it directly above the first band. This will be used to measure background
- Repeat this for every band on the gel (one box for the band, one box for background)
- Record this data along with gel picture on the wiki
- To determine p7308 concentration, use background-subtracted value for each volume. Scale each unknown concentration against the control (44 nM) according to the ratio of background-subtracted intensity for the band that looks closest in intensity
- For example, if the band in lane 1 (3 μL, 44 nM) had an intensity of 1000, and the band in lane 5 (3 μL, ?? nM) had an intensity of 900, then we would record 900/1000 * 44 = 39.6 nM as the estimated concentration for that lane. Repeating for each lane should give you 3 data points, which you can average (throwing out any obvious outliers).
0.5x TBE, 11 mM MgCl2
| Lane | Contents | Loading Buffer |
| 0 | 1kb DNA ladder (5 μL) | |
| 1 | p7308 060323, 44 nM (3 μL) | AGLB (2 μL) + dH2O (9 μL) |
| 2 | p7308 060323, 44 nM (6 μL) | AGLB (2 μL) + dH2O (6 μL) |
| 3 | p7308 060323, 44 nM (9 μL) | AGLB (2 μL) + dH2O (3 μL) |
| 4 | p7308 060522, 1:2 dil (1 μL) | AGLB (2 μL) + dH2O (11 μL) |
| 5 | p7308 060522, 1:2 dil (3 μL) | AGLB (2 μL) + dH2O (9 μL) |
| 6 | p7308 060522, 1:2 dil (6 μL) | AGLB (2 μL) + dH2O (6 μL) |
| 7 | p7308 060522, 1:2 dil (9 μL) | AGLB (2 μL) + dH2O (3 μL) |
| 8 | p7308 060522, 1:2 dil (12 μL) | AGLB (2 μL) |
Magnetic Streptavidin Bead "Protection" Assay Gel
- Gel:
| Lane | Component | Amount |
| 1 | water control | 20uL |
| 2 | c5.0.8(b) | 20uL |
| 3 | c5.0.E(b) | 20uL |
| 4 | c5.0.F(b) | 20uL |
| 5 | 1kb+ ladder | |
| 6 | p7308 |
-
2% agarose, EtBr, 10mM MgCl2
- Gel Results:
- Faint band in lane 4 (c5.0.F(b)) was seen (unclear in image file).
- Since only ~40% of a 40uL reaction was used, this is good reason to try again with full reactions worth of nanostructures.
- This is also a bit baffling - F(b) is supposedly inside biotinylated, and shouldn't have been retained by the streptavidin beads at all. If a similar band had been seen in lane 3 (c5.0.E(b), outside biotinylation) this result would be understandable - implying that protection of the biotinylated sites did not occur - but as it is, the best conclusion is that the tubes must have been mixed up at some point, or that the working stocks were mixed up.
- Conclusion: Redo with higher reaction volume.
- Faint band in lane 4 (c5.0.F(b)) was seen (unclear in image file).
Magnetic Streptavidin Protection, Take 2
(Generally the same protocol from Monday 8.14 was used, but some alterations were made based on recalculations and necessary volumes.)
- Folding:
- Folded at normal 10mM MgCl2, 1x oligo conditions two 40uL reactions each of c5.0.E(b) and c5.0.F(b) using FOLDINGD.
- Recalculations:
BIOTINYLATED BOXES
------------------
1 40uL-rxn = (5 biotin sites)*(9ul of p7308, the limiting reagent in folding reactions)*(44picomoles/1uL = 44uM, concentration of p7308)
= 1680pmoles of binding sites/rxn
= 336pmoles of nanoboxes/rxn (important because we might expect each streptavidin-cum-bead to only be able to fit in the available space around one nanobox)
FREE-FLOATING BIOTINYLATED OLIGOS
---------------------------------
1 40uL-rxn = (16uL of working stock oligos)*(5ul of the biotinylated-oligos-pre-working stock/200uL of pre-working stock in the working stock)*(10picomoles of each biotinylated oligo/1uL of pre-working stock = 10uM)*(5 biotinylated oligos in the c5.0 design)
= 20pmoles of binding sites/rxn, then minus the 1:10 which goes into making the actual biotinylated boxes
= 36pmoles of binding sites/rxn
---> Thus, we must have enough beads to bind significantly >36pmoles of biotin, because then we can be sure that, even if every biotinylated oligo was bound, there'd still be the capacity for boxes to be bound.
---> BINDING CAPACITY OF BEADS: 2pmoles/uL of beads. Thus, for total binding of the 1716pmoles (=1680pmoles+36pmoles) of biotin in each reaction, we would need 858uL of beads. Since the beads cost ~$200 for 5mL, this seems an unwise amount to use.
---> But 200uL of beads, with consequent binding capacity of 400pmoles, will (worst-case scenario) bind all 36pmoles of free-floating biotinylated oligos AND 365pmoles of nanobox sites, which is approximately 1/5-1 full reaction worth of nanoboxes, depending on the bead:boxes-bound ratio.
- Protocol:
1. Incubate:
200uL beads
160uL 1x folding buffer
40uL test solution
TEST SOLUTIONS:
--------------
a) 6hb (to show that plain unbiotinylated structure won't bind streptavidin)
b) 10uL of biotinylated oligos (c5.0.8(b)) + 30uL H2O (=500pmoles, comparable to reactions' binding capacity
c) c5.0 E(b) (outside biotinylation) - unpurified, because was folded at 10mM MgCl2 and an appropriate PEG precipitation percentage had not been determined for this folding condition
d) c5.0 F(b) (inside biotinylation)
2. Mix by thorough trituration
3. Pellet by drawing magnet down to bottom of tube (~ 1 minute using the MagnaRack)
4. Remove supernatant to separate tube (all supernatant from these washes were collected in one tube per reaction)
5. Add 100uL 1x folding buffer
6. Repeat steps 2-5 three more times.
7. Trypsinize by adding to pellet:
5uL trypsin (1mg/mL)
195uL 1x folding buffer
8. Incubate overnight @ 37 degrees C
9. Pellet (1 minute using the MagnaRack) and remove the final-supernatant to a clean tube.
10. Speedvac down:
the 4 wash-supernatants for each of the test solutions
the 4 final-supernatants for each of the test solutions
until the final-supernatants are dry (~1hr @ 45C). Reconstitute to 30uL, to fit into gel wells.
11. Run 30uL of each supernatant on 2% agarose gel (10mM MgCl2) for 1hr at 80V.
- Steps 9-11 were done Wed 8.16.
