Generic sample from Microctonus aethiopoides
Microctonus aethiopoides
     Dissection of venom glands:     Female adult wasps, after wetting and sterilisation for 20 seconds in 70% ethanol, were held in a PBS solution in a watch glass and the ovipositor pulled from the rear of the abdomen using fine forceps under a stereomicroscope. The venom gland, venom reservoir and ovaries remained attached to the ovipositor and they could then be identified. The venom gland was removed using fine forceps and placed immediately in 30 microliters of Trizol (Invitrogen, Carlsbad, CA). The venom gland reservoir was also removed and immediately placed in 5 microliters of  4x NuPAGE LDS sample buffer (Invitrogen). In order to preserve the integrity of the samples any dissection which took longer than 2 minutes from the time of ovipositor removal until the gland or reservoir were in the appropriate buffer was abandoned.          RNA extraction:     To prepare RNA between 30 and 40 dissected glands were placed in 30 microliters of Trizol and processed according to the manufacturers instructions (Sigma) in a small sterile 0.6 ml polypropylene     Eppendorf tube. Sterile glycogen, as suggested by the manufacturer, was added at the final ethanol precipitation step to ensure that the very small amounts of purified RNA were precipitated and     could be collected by centrifugation. The RNA from the extraction was dissolved and stored in sterile, DEPC treated, distilled water at -70 degrees C.          Pyroseqencing:     The DNA used for sequencing was 6 micrograms amplified cDNA derived from venom RNA prepared from M. aethiopoides venom glands dissected and purified as described above. The SMART cDNA preparation protocol (Clonetech) was followed according to the manufacturers instructions using 3 microliters of RNA solution as starting material and 26 cycles of amplification. To obtain the quantity of cDNA required for sequencing (6 micrograms) the cDNA protocol was repeated three times and the amplification products were pooled.     