imaging 2:
dna paint
CLASS ASSIGNMENT
LAB TASK
This protocol is for folding a six-helix bundle with twelve 3prime 21mer handles of sequence GCTCTGCAATCAACTTATCCC on the upstream side, and twelve 3prime 21mer handles of sequence CGTCCCCTTTTAACCCTAGAA on the downstream side.
There are four biotins interspersed along the length for capture on streptavidin-coated glass slides.
Pipetting Scheme
1. Hydrate staple strands for P0–P2 (IDT wells shipped dry)
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Assume an average of 10 nmol per well for P0, P1 and 70 nmol per well for P2
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Add 100 μL water to each DNA-containing well (see below) to achieve an nominal average concentration of 100 μM for P0, P1 and 250 μM for P2
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P0A01–P0H12
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P1A01–P1F04
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P2A01–P2C04
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Spin down plate
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Incubate at room temperature for 10 min
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Seal, vortex, spin down plate
2. Generate pre-stocks (pipet 10 μL from each well from P0, P1 or 4 μL from each well from P2)
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.A_PS: P0A01–P1F04: light gray staple strands (160 total)
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.B_PS: P2A01–P2C04: red and blue handle-bearing staple strands, purple biotinylated staple strands (28 total)
3. Generate working stock at 500 nM per strand
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.0_WS: 160 μL .A_PS + 1
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1.2 μL .B_PS + 28.9 μL water (200 μL total)
4. Aliquoting and distribution to teams
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Add protocol steps here
5. Folding cocktail
Ingredient
50 mM Tris, 10 mM EDTA pH 8.0
100 mM MgCl2 stock
p8064 100 nM stock
water
.v0.0_WS stock (500 nM per strand)
Total volume
1x volume in μL
5
5
5
25
10
50
Master cocktail 2x volume in μL
10
10
10
50
20
100
Lane
0
1
2
Contents
2 log ladder
p8064 100 nM
6025-18h, 10 mM MgCl2, 6025-18h
Volume sample in μL
5
15
15
Master cocktail 2x volume in μL
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5
5
Volume sample
30
30
30
6. Thermal ramps
6025-18h: 80°C 15 min → 60°C to 25°C over 18h
Results
1. Gel analysis
2% agarose gel; 1x TAE buffer, safeguard coloring; 120V for 45 min at RT
Lab notes
The protocol for gel electrophoresis can be found here. How to do this is also explained in HTGAA class 2.
First, the TAE buffer and agarose are mixed and poured in the gel electrophoresis chamber. The safeguard coloring is also added to the TAE buffer solution (300 ml to fill the entire chamber). This is usually mixed in the gel before it sets but it can also be added later, in the liquid buffer that fills up the entire chamber.
For the gel electrophoresis, it is important to hook up the power source correctly. DNA has a negative charge, so make sure the red wire is connected to the end of the gel. The DNA runs from the negative (black wire) to the positive side (red wire). If this is done incorrectly, the DNA will run off the gel in the wrong direction. Turn the power source on at 120 Volt and make sure to shut it off before inserting the DNA.
In every other slot, 30uL of each of the following mix was added:
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Assembled DNA ladder
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DNA origami
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Single Stranded (SS) DNA
When all slots are filled, the electricity was turned on. We waited for 45 min
to an hour. The results can be seen in the image.
2. TEM analysis
In John's lab!
