How to accurately pipette genomic DNA

[mdalessio]

Hey everyone,

How do you pipette genomic DNA samples accurately? I have tried letting them sit at 65 C to encourage their equal distribution in the sample. For the XT kit it only wants 1 ng of DNA, and I have a hard time trusting that my 1 ul sample will definitely contain that 1 ng.

[ScottC]

We just use good pipetting technique... nothing special. For NexteraXT where we attempt to use 0.8ng, we carry out a serial dilution and measure by fluorometry (Qubit, DNA HS kit). We take a dilution that is in a few fold difference of our target, and carefully dilute that without using very small volumes (which are likely to be more inaccurate). When we have to use small volumes, we use unfiltered tips.

[james hadfield]

ScottC's right. Good pipetting, large volumes (>5ul) and serial dilution till you get to the required concentration. Don't even try and use Nanodrop!

Hopefully Illumina et al will move to a bead based normalisation before Nextera protocols to help users out here.