Hi everyone. I'm performing DNA extractions for pools of ~10 individual Daphnia magna (water fleas) using the Qiagen QIAamp DNA Micro Kit. I use a pestle to homogenise the Daphnia before entering them into a 2.5hr pK digestion step. The rest of the protocol I follow to the letter. I then re-elute the DNA by following the QIAamp Cleanup Protocol.
The largest DNA fragments I've been able to recover (according to a gel run) is ~1000bp, but the majority of fragments are 200-500bp in every run I've tried. I need larger fragments for PE sequencing, and was wondering if anyone can think of anything I can try to get longer eluted DNA fragments?
So far I have tried:
1) Pipetting mixtures up and down, rather than vortexing - this seems to have produced smaller average fragment lengths
2) Homogenising the Daphnia before adding ATL buffer vs adding the ATL and then homogenising - this makes no difference
3) Testing frozen Daphnia vs freshly caught Daphnia in case of degradation - this makes no difference
4) Testing different kits - very poor returns using Zymo Quick DNA Mini Prep Plus kit and Qiagen DNEasy kit. In fact, nothing showed on the gel at all for these kits!
Any advice will be greatly appreciated!
Ian
I solved the problem - it turns out the QIAamp kit columns elute RNA as well as gDNA. This should not happen, according to the kit. But then the pH levels of the buffers are not as described in the Qiagen kit handbook either (I used dip sticks to test multiple boxes of the same kit), so I wonder if this could be a contributing factor.
At any rate, I added a step AFTER the pK digestion and BEFORE the addition of Buffer AL and EtOH. I added 4uL of 100mg/mL RNase A, vortexed and incubated at room temperature for 2 mins. For my small samples, this resulted in poor Nanodrop readings and no visible smears on a gel, but QC by our sequencing service provider showed that there was sufficient DNA of sufficient length and purity for our needs.
The 'take home' message is don't necessarily trust that kits work precisely as they claim to!
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