Does anyone have SDS based DNA isolation protocol from Aedes aegypti mosquito larvae or larvae of any related organisms? I have to isolate DNA from freshly emerged mosquito larvae and older. I have tried this protocol once:
1: grounded the larvae in TE pH 8.0
2. Added 10 uL of 10mg/mL RNase A. Incubated at 37'C for 30 min
3. Added 25 uL of 10mg/mL Proteinase K. Incubated at 55'C for 15 min.
4. Added 30 uL of 20% SDS. Incubated at 60'C for 30 min.
5. Continued with chloroform:isoamyl alcohol (24V:1V) extraction.
6. Resuspended in 30uL of TE buffer.
When I used picodrop to quantify the amount of DNA, I had low yield. Moreover, the absorption was funny looking. I had large flat peak ranging from about 200-500 nm. Does anyone have guess on what might have happened? I appreciate the help in advance.
we use an adapted protocol employing the miniprep columns of Macherey_Nagel for genomic extraction from Ae aegypti adults. Although it shears the DNA this is okay for the applications we use it for (and its v quick). For applications when we need the intact genome we use a protocol involving crushing the material in Bender buffer, overnight Proteinase K incubation then a Phenol-Chloroform followed by precipitation. I can provide further details in you are interested. We have used it for gDNA extraction from all stages of Ae aegypti.
From looking at your protocol I think the Proteinase K (+RNase A) incubation needs substantially longer. Also - what do you qualify as a low yield? - what are you hoping to use the material for?
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