I was trying to sequence a chromosome. I purified it with short read eliminator (>10kb) and initial quantification was above 100 ng I used the LFB long fragment buffer of nanopore, my nanodrop regarding on qubit is very low. Says out of range. Can someone suggest something to rectify this?
Hi Ankush, I have been optimizing nanopore protocols the last few months. What I can say is that the nanopore protocols seem to decrease the incubation timings recomended for both ligation enzymes and AMPure beads.
What helped me (I used the ligation sequencing kit):
- I have increased the End-Prep reaction timings as NEB suggested: 30' at 20º -- 10' at 65º.
-I have increased the barcode ligation to 40' at room temperature instead of 10'.
-I have increased the beads incubation times a bit (like waiting 5 minutes instead of "until the eluate is clear")
-I have increased to 10' the elution time after the washes AND 37º instead of room temperature.
Hope this helps!!
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