I'm working on making a custom ss probe set double stranded, so I can sequence them on an oxford nanopore. After my initial investigation, I'm wondering how I should go about this. I first tried to add a hexmer_tag primer, then extending my fragment with a klenow, NEBuffer2 and dNTP mix. Some things to note about my first attempt:

1.) my probes are 120bp long, ~ 84.5ng/ul

2.) the IDT primer tag we ordered is below (30bp long)

Hexamer_tag: ATGTACGACAATATCGCTTAGTCACCCTTGNNNNNN

Hexamer_tag complement: /5Phos/CA AGG GTG ACT AAG CGA TAT TGT CGT ACA T

Both tubes were resuspended in H2O based on IDT's calculating buffer volume when resuspending oligos

3.) I used the ligation NEBCalculator to estimate the amount of primer (insert DNA) to add to my probes, at a 3:1 ratio

4.) I combined the two hexamers pieces at 95˚C for 30 seconds then added them to the aliquot of probes

5.) I used NEB's A-Tailing with Klenow Fragment (3'-->5' exo-) to extend the sequence.

Purified Blunt DNA: 1-5 μg

NEBuffer 2 (10X): 5 μl

dATP (10 mM): 0.5 μl (0.1 mM final)

Klenow Fragment (3´→ 5´ exo–): 3 μl

Sterile H2O: variable

Total volume: 50 μl

Incubate in a thermal cycler for 30 minutes at 37°C.

The eluate was cleaned with a 2x bead clean-up.

6.) The final elution after the bead clean up had a concentration of 1.3ng/ul (total mass: 65ng).

7.) I ran the material on a lab chip (see attached images) but found the sizes of my fragments at ~120bp.

It appears the primers didn't attach and I'm wondering if my probes have any blockers that would hinder the attachment of the primer? Our probes are 5'only biotinylated. Could I use an NEB DNA repair kit to clean up an end of the probe THEN running a Klenow reaction? If you have any other ideas on how I could investigate and make the ssprobe ds, I'm open ears.

Thanks