Lambda control library problem

[mkdir]

We have had happy experience with 454, pgm and illumina libraries. However, when we followed the minION Lambda DNA control library protocol, we can only get 25 ng DNA instead of the expected 250 ng.

First of all, the provided lambda DNA cannot be sheared to ~ 6kb to 8k even at 16,000 X g using g-tube. This is unusual based on our experience and Covaris protocol. The final sizes of the sheared DNA are around 12-15 kb. The support team told us no need to worry about the size...... After end-prep process, the yield of DNA is 300 ng instead of the expected 700 ng (30% instead of 70%). The final yield of the library is even worse, around 25 ng (8.3% instead of 35% ).

Two posts in the Nanopore community showed that the provided lambda DNA cannot be sheared to 8 kb. They got 16Kb. But nobody replied to those two posts...


Can we load the pooled ~ 60 ng library to minION? Any suggestions will be helpful. Thank you very much in advance!

[Ola]

Sure, you can load whatever you have. 60 ng should be fine. In case you get poor yields (i.e have many single pores but few in strand) you can just stop the run and add a new library. I have not sheared lambda with g-tubes but used restriction enzymes instead with good results.

[wdecoster]

Shearing using g-tubes has never been a problem here...
Did you quantify the sample also after shearing?

Steps in which you commonly lose much material are purification steps, it might help to elute from the beads for a longer time on elevated temperature. We commonly do 15 minutes on 37 degrees. Note that longer DNA will be tougher to elute.

[mkdir]

Ola, Thank you very much for the information.

Sure, you can load whatever you have. 60 ng should be fine. In case you get poor yields (i.e have many single pores but few in strand) you can just stop the run and add a new library. I have not sheared lambda with g-tubes but used restriction enzymes instead with good results.

[mkdir]

Hi wdecoster, thank you very much for the suggestions. We didn't quantify the samples after shearing by nanodrop or other quantification kit. But we tested the sizes of the sheared lambda DNA with different g-forces by running agarose gel. When compared to the brightness of the separated bands of the ladder, the brightness of the sheared lambda DNA bands look good. We don't think that we lose DNA during shearing process.

We did 10 min incubation at 37 degree, which eluted ~ 25 ng. We further did 10 min incubation with another ELB at 66 degree, which eluted ~ 20ng.

Shearing using g-tubes has never been a problem here...
Did you quantify the sample also after shearing?

Steps in which you commonly lose much material are purification steps, it might help to elute from the beads for a longer time on elevated temperature. We commonly do 15 minutes on 37 degrees. Note that longer DNA will be tougher to elute.