Single-stranded Low Complexity DNA Curtains

Contents

1. Anneal biotinylated primer and template
2. A new method of oligonucleotide annealing
3. Rolling Circle Reaction
4. References

1. Anneal biotinylated primer and template

• IF238: Biotinylated TC primer
• IF239: Phosphorylated AG template

Ligation Reaction Set-up:

• 5 uL 10x Ligase Buffer (NEB stock)
• 2 uL IF239 (5 uM stock)
• 2 uL IF238 (4.5 uM stock)
• 40 uL H2O

1. Heat to 75C for 5 minutes in the thermocycler.
2. Cool to 4C at a rate of -1C/min.
3. Add 1 uL of NEB T4 DNA Ligase.

Above papers say to let sit at RT for 5 hours. I have done incubations between 3-5 hours and have not noticed a significant difference.

If you are wondering why there is a different concentration for the two oligos… we want to limit the amount of free biotinylated oligos (not bound to 239) that will bind the straptavidin on surface.

2. A new method of oligonucleotide annealing

Look here for setting up ssDNA Curtains preparing_ssDNA_curtains

Ligation Reaction Set-up:

• 5 uL 10x Ligase Buffer (NEB stock)
• 2 uL IF239 (100 uM stock)
• 1.8 uL IF238 (100 uM stock)
• 40.2 uL H2O

1. Heat to 75C for 5 minutes in the thermocycler.
2. Cool to 4C at a rate of -1C/min.
3. Add 1 uL of NEB T4 DNA Ligase.
4. Incubate at RT for ~5 hours. Then put in the 4C indefinitely.

3. Rolling Circle Reaction

Rolling Circle Reaction Set-up:

• 1 uL Ligation Reaction (above) - (Dilute 20x if using the new Ligation protocol)
• 10 uL 10x Phi29 NEB Buffer
• 2.5 uL 10 mM dTTP
• 2.5 uL 10 mM dCTP
• 1 uL 1 mg/mL BSA (100X BSA)
• 1.5 uL of homemade Phi29 DNAP (currently using the pET19 made WT phi29 - pIF376)
• 37 uL H2O

1. Incubate ON FLOW CELL at 30C for ~20 minutes. This is the time I have tested and it gives long ssDNA molecules. You can optimize time for ssDNA length as you wish.

4. References

Adapted from:

• PubMed 24792418
• PubMed 22956981