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Folding 5.0.A and 6.0
Design 5
- Make working stock c5.0.A
Working Stocks
|
Description
|
Pre-Working Stocks
|
Water
|
Total
|
c5.0A |
no latches, no aptamers |
1 (86 μL), 2 (49 μL), 3 (49 μL), 4 (2.5 μL), 5 (2.5 μL), 6 (1.5 μL), 7 (1.5 μL) |
8 μL |
200 μL
|
Design 6
- Mixing pre-working stocks (c6.0.1, c6.0.2, c6.0.3, c6.0.4, c6.0.5)
- To mix pre-working stocks from plates, pipet 10 μL of each of the appropriate oligos into 1.5 mL tubes.
- Mix working stocks c6.0.A, c6.0.B, c6.0.C
Pre-Working Stocks
Description
|
Plate Locations
|
Total
|
c6.0.1: core barrel oligos |
box_v6_A; box_v6_B rows A-D (1-12), row E (1-9) |
153
|
c6.0.2: barrel oligos @ outside aptamers -aptamers |
box_v6_B row E (9-12), row F (1-12) |
15
|
c6.0.3: barrel oligos @ inside aptamers -aptamers |
box_v6_B row G (1-6) |
6
|
c6.0.4: barrel oligos @ outside aptamers+ligand |
box_v6_B row G (7-12), row H (1-12); box_v6_C row A (1-11) |
29
|
c6.0.5: barrel oligos @ inside aptamers+ligand |
box_v6_C row A (12), row B (1-12), row C (1-5) |
18
|
Working Stock
Stock ID |
Experiment |
1 |
2 |
3 |
4 |
5
|
c6.0A |
no aptamers |
x |
x |
x |
- |
-
|
c6.0B |
in aptamers |
x |
x |
- |
- |
x
|
c6.0C |
out aptamers |
x |
- |
x |
x |
-
|
Working Stocks
|
Description
|
Pre-Working Stocks
|
Water
|
Total
|
c6.0A |
no aptamers |
1 (153 μL), 2 (15 μL), 3 (6 μL) |
26 μL |
200 μL
|
c6.0B |
in aptamers |
1 (153 μL), 2 (15 μL), 5 (18 μL) |
14 μL |
200 μL
|
c6.0C |
out aptamers |
1 (153 μL), 3 (6 μL), 4 (29 μL) |
12 μL |
200 μL
|
Quantify p7308
- Need to quantify p7308 to have enough scaffold to fold everything.
- Pour 2% agraose, 11 mM MgCl2 gel
- 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).
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 μL) |
AGLB (2 μL) + dH2O (11 μL)
|
5 |
p7308 060522, (3 μL) |
AGLB (2 μL) + dH2O (9 μL)
|
6 |
p7308 060522, 6 μL) |
AGLB (2 μL) + dH2O (6 μL)
|
7 |
p7308 060522, (9 μL) |
AGLB (2 μL) + dH2O (3 μL)
|
Folding protocol
- Folding c5.0.A, c6.0.A, c6.0.B, c6.0.C - 24 tubes ea. * 40 μL per tube = 960 μL of each design
- since new scaffold still needs to be quantified, will use older scaffold (44nM)
- Each rxn:
16 ul oligos (from working stock)
9 ul p7308
11 ul H20
4 ul 10x folding buffer
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.
- Nanostructures:
- using 30mM MgCl2, 1x oligos (seems to work best given previous results).
- designs: c5.0.A, c5.0.C, c5.0.D key
- PEG: 8%, 10%. Total volume in each is 100 μL
40 μL 20% PEG
10 μL 5M NaCl
10 μL water
40 μL Nanostructures (add last)
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