Single-stranded DNA Curtains
Contents
Section titled “Contents”1. Anneal biotinylated oligo to M13 ssDNA
Section titled “1. Anneal biotinylated oligo to M13 ssDNA”- Combine:
- 20 ul of M13mp18 ssDNA (N4040S, NEB) (Cf: 40 nM)
- 1.5 ul of 1 uM of biotin-primer (Cf: 33.3 nM)
- 5 ul of 10x annealing buffer (Cf: 1x)
- 23.5 ul of H2O
- Heat the mixture at 95˚C for 10 min and slowly cool down to RT.
No further purification is necessary because M13 ssDNA concentration is higher than that of the biotin-primer.
Aliquot and store at 4˚C for short-term storage (< 1 month). For long-term storage, -20C is recommended.
Sometimes, the annealed M13&biotin-primers is suddenly degraded. We do not know the reason. Just throw out that aliquot and go to the next sample.
2. Rolling Circle Replication
Section titled “2. Rolling Circle Replication”2.1 Reaction
Section titled “2.1 Reaction”Rolling circle reaction
| Component | Amount | Cf |
|---|---|---|
| 5x Reaction buffer | 10 ul | 1x |
| 10 mM dNTPs | 1 ul | 0.2 mM (10 nmol) |
| 15 nM M13 & bioprimer | 1 ul | |
| Φ29 polymerase | 1 ul | 100 nM |
| D.W. | 37 ul |
Total volume: 50 ul
- Incubate at 30˚C for 25 min. Note: 25 min is not critical. But the longer time you incubate, the longer ssDNA you will get.
- After the rolling circle reaction is done, add ~850 ul of BSA-buffer (with 1 mM DTT) to the reaction.
- Inject into the flowcell coated with streptavidin. Use same day, the long ssDNA does not store well.
2.2 Buffers
Section titled “2.2 Buffers”5x Reaction buffer
- 250 mM Tris-HCl (7.5)
- 20 mM DTT
- 50 mM Ammonium Sulfate
- 50 mM MgCl2
| Component | Volume of Stock | Cf |
|---|---|---|
| 1 M Tris-HCl (7.5) | 12.5 ul | 250 mM |
| 1 M DTT | 1 ul | 250 mM |
| 1 M Ammonium Sulfate | 2.5 ul | 50 mM |
| 1 M MgCl2 | 2.5 ul | 50 mM |
| D.W. | 31.5 ul |
Total: 50 ul
3. Preparing the flowcell
Section titled “3. Preparing the flowcell”- Prepare the flowcell as you would for any double-stranded DNA experiment.
- Add 0.5 ul of 10 uM GFP-RPA to 20 ml of BSA buffer supplemented with 1 mM DTT (Cf: 0.25 nM).
- Connect the GFP-RPA buffer to the flow system.
- Use 30 ml syringe.
- Connect the flow cell with the flow system.
- Flow the RPA-GFP buffer at 1 ml/min for 15 min. During this injection, check the single-stranded DNA curtains.
- If you wish to remove RPA:
- Inject 7 M Urea after 5 min, which will help remove aggregates of RPA and elongate ssDNA molecules.
The surface of flowcell must be good to get proper ssDNA curtains. If the surface is bad, there are many bright spots on the surface, which seem to result from the aggregated RPA-GFP.
4. References
Section titled “4. References”Adapted from: PubMed 22950646