I'm attempting to extract DNA from a single retrix from a small sparrow. I have upwards of 200 samples but only one feather per individual. The feathers have no visible blood spots on them.
I've tried phenol chloroform, a pellet method developed by De Volo et al (2008), and magnetic beads. I've also tried using glycogen with no luck. On average, my samples yield less than 0.09 ng/uL.
I typically use about the first centimeter or so from the base of the rachis, which is just above the super umbilicus.
I have yet to try any column based methods.
Are there any suggestions or methods that might be helpful? My goal is to get just enough DNA for whole genome amplification, so about 1-5 ng/uL.
Thank you.
With column based method, you would certainly not get any DNA in your situation. There is very less recovery in column based method, as washing losses a lot if DNA.
Hi, for HMW DNA indeed I wouldn't recommend column based protocols/kits. Even if you manage to get enough DNA, it would be probably sheared. I have had used phenol/chloroform method on plant-parasitic nematodes with no vortexing, very careful pipetting and mixing only by inversion. Ensure phenol is fresh (less than 6-month old) since it can be oxidised. and do not expose DNA on T>65 for more than an hour. After adding phenol and phase separation, pick up the aqueous solution and then add elution buffer to the rest of the phenol phase, centrifuge again to recover more DNA. At the end, elution on Tris overnight at RT can increase signficantly the DNA quantity at the end.
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