Preparation of Nanobot (DNA attached beads)
1. We took 20 µL of streptavidin beads from stock into a 500 µL tube.
2. After vortex of the tube, we centrifuged supernatant and re-suspended in BW buffer (Washing buffer) 100 µL.
3. We washed three times with 100 µL of BW buffer (Centrifuge and re-suspension).
4. In order to anneal strands, we put pairs of linker DNAs written as below on 95°C for 10 minutes in other tubes, then cooled at room temperature for 10 minutes.
-mix1: D1 unit1 biot 100 µM 3 µL + D1 unit2 100 µM 3 µL
-mix2: D2 unit1 biot 100 µM 3 µL + D2 unit2 100 µM 3 µL
5. We mixed beads solution and strands solution as following.
-D1: 24 µL of TE buffer + 6 µL of mix1
-D2: 24 µL of TE buffer + 6 µL of mix2
6. We washed D1 and D2 as following:
-100 µL of BW buffer two times.
-100 µL of SB buffer two times.
Finally we suspended D1 and D2 in SB buffer.
Preparation of chamber
1. Cut parafilm into rectangular slices
2. Put the slices on a microscope slide
3. Put cover glass on the slices
4. Heat the glasses to fix
5. Put samples in lanes respectively
6. Apply glue on the edge of the cover glass in order to prevent the samples from evaporating
7. Put the chamber on thermo plate and observe it with microscope
Protocol
Experiment of BIT
| 1. MasterMix | |
|---|---|
| PPmix-dNTP | 25% |
| dNTP | 4% |
| α to α 1uM | 1% |
| BSA9000S | 1% |
| RecJ | 1% |
| Bst | 1.5% |
| NB.Bsml nickase | 4% |
| EvaGreen | 5% |
| Molecular beacon | 2.5% |
| Tween 1% | 4.5% |
| TE buffer | full-up |
| 2. PutTrigger | |
| drainα:20nM | |
| Sample1 α0pM | Mastermix + TE buffer+drainα20nM |
| Sample2 α100pM | Mastermix + α 100pM+drainα20nM |
| Sample3 α1nM | Mastermix + α 1nM+drainα20nM |
| Sample4 α10nM | Mastermix + α 10nM+drainα20nM |
| drainα:0nM | |
| Sample5 α0pM | Mastermix + TE buffer |
| Sample6 α100pM | Mastermix + α 100pM |
| Sample7 α1nM | Mastermix + α 1nM |
| Sample8 α10nM | Mastermix + α 10nM |
| 1. MasterMix | |
|---|---|
| PPmix | 25% |
| D1 stock | 10% |
| D2 stock | 10% |
| Tween 20 | 0.05% |
| MQ | full up |
| 2. PutTrigger | |
| Sample1 | Mastermix + Linker 10uM |
| Sample2 | Mastermix + Linker 1uM |
| Sample3 | Mastermix + Linker 0.1uM |
| Sample4 | Mastermix + MQ |
| 1. MasterMix | |
|---|---|
| Ppmix-dNTP | 25% |
| dNTP | 4% |
| α 1uM | 1% |
| drainα1uM | 2% |
| BSA9000S | 1% |
| RecJ | 1% |
| Bst | 1.5% |
| NB.Bsml nickase | 4% |
| EvaGreen | 5% |
| Tween 1% | 1.5% |
| TE buffer | full-up |
| 2. PutTrigger | |
| Sample1 α to Linker (-) | Mastermix |
| Sample2 α to Linker (-) | Mastermix |
| Sample3 α to Linker (+) | Mastermix + α to Linker 10nM |
| Sample4 α to Linker (+) | Mastermix + α to Linker 10nM |
| 1. MasterMix | |
|---|---|
| Ppmix-dNTP | 25% |
| dNTP | 4% |
| α to α 1uM | 3% |
| α to Linker 1uM | 1% |
| D1 beads | 10% |
| D2 beads | 10% |
| drainα1uM | 2% |
| BSA9000S | 1% |
| RecJ | 1% |
| Bst | 1.5% |
| NB.Bsml nickase | 4% |
| EvaGreen | 5% |
| Tween 1% | 4.5% |
| TE buffer | full-up |
| 2. PutTrigger | |
| Sample1 α800pM | Mastermix + α 800pM |
| Sample2 α700pM | Mastermix + α 700pM |
| Sample3 α600pM | Mastermix + α 600pM |
| Sample4 α500pM | Mastermix + α 500pM |
| Sample5 α400pM | Mastermix + α 400pM |
| Sample6 α300pM | Mastermix + α 300pM |
| Sample7 α200pM | Mastermix + α 200pM |
| Sample8 α100pM | Mastermix + α 100pM |
| Sample9 α0pM | Mastermix + TE buffer |
Materials
Streptavidin Coated Microspheres (BAN Bangs Labratories, Inc. Catalog number: CP1004)
Evagreen®, 20X in water, Unlabeled (BTI, Catalog numbe: 31000)
NB.BsmI (BioLabs, Catalog number: R0706)
Bst 2.0 WarmStart DNA Polymerase (BioLabs, Catalog number: M0538)
Nt.BstNBI (BioLabs, Catalog number: R0607)
RecJtt (Homemade)
DNA strands (Integrated DNA Technologies, US)
| Nanobot strands | |
|---|---|
| D1 unit1 | biot/*C*C*CACAGGCACGCTCAACGCTAACC/Cy5 |
| D1 unit2 | C*G*T*CGAATGTAG GGTTAGCGTTGAGCGTGCCTGTGGG/phos |
| D2 unit1 | Cy3/C*C*C*ACAGGCACGCTCAACGCTAACC/biot |
| D2 unit2 | G*G*G*TGTCCGTGCGAGTTGCGATTGG TCG TTATCGCATCC/phos |
| Bifurcation | |
| α | CATTCAGGATCG |
| α to α | CGATCCTGAATG CGATCCTGAA |
| drain α | T*T*T*TT CGATCCTGAATG |
| α to linker | ACA TCG CAT CCC GTC GAA TGC GAT CCT GAA |
| linker | CATTC GACG GGATGC GATGT |
| molecular beacon | BHQ1/*A*T*TCAGAATGCGATCCTGAAT/Cy5 |
Stations
CFX96 (BIO-RAD, Catalog number: 1855196J1)
Nikon Eclipse Ti2 (Nikon)