double DNA extraction?

[bensen]

hi everyone. For GBS-analyses i need high quantity and quality gDNA of around 400 samples of a violet species (dried material). When i´m using the Qiagen DNeasy plant kit 96 and elute with 50ul TE i get between 10 and 30 ng/ul measured with a nanodrop. However as the nanodrop often seems to overestimate DNA concentration, my yields might be to small for the analyses (optimum 50 - 100ng/ul).

To optimice the DNA concentration i thought of two different ways ...

1) making two parallel DNA extractions of the same sample, elute each with 100 or 200ul TE, combine them and finally make an ethanol precipitation.

2) making two parallel DNA extractions of the same sample, elute the first with 50 or 100ul TE, use the resulting elution from the first sample again for the second sample and in this way increase the final concentration.

In the moment i prefer variant 2) as it is easier, much faster and includes less steps than variant 1) (-> 400 ethanol precipitations in single 2ml tubes). What do you think, is this worth doing or do you have any other suggestions how to concentrate the DNA in the 96 wells?

Thanks for any answers,
Ben

[JamieHeather]

Probably also worth trying a more accurate, fluorometric based DNA quantitation; you might not need to be fiddling with your extraction protocols.

[JeremyDay]

First have you tried eluting off the columns with smaller volumes (25x2)? And use prewarmed Elution buffer.

Depending on your lab, it may be a large investment, but quant-it picogreen is pretty sensitive (50pg/ml I think). It is $400/ml (2500 samples) , about $0.15 per sample, so $0.45 if done in triplicates. You can setup a standard curve, and use this in 96 well plates with a plate reader such as spectramax. If you are already using multichannel pipetters and using the 96-well kit, you could analyze 400 samples much faster than nanodropping them.

If you have $400 to spend, you could buy a 96 well magnet from Alpaqua (there may be cheaper options, I use this for a robot too).
Then how about: MagAttract 96 DNA Plant Core Kit from Qiagen also. There is a possibility you could elute with smaller volumes since it is off beads.

I am not sure how much of the workflow you are doing, but Ampure Beads can be homemade and work for many applications, and since you are doing a lot of samples, there is a chance a 96 well magnet could be handy down the road.

I know these options cost $$, but the time you could save is priceless