A 241 bp PCR fragment of SeNPF with a homologous sequence of L4440 was amplified by the corresponding primer pairs (Table 2 ), and then cloned into the linearized vector L4440, amplified by the corresponding primer pairs, by the Cloning Kit (ClonExpress®II One Step Cloning Kit, Novoprotein Sicientific Inc., Shanghai, China). L4440-SeNPF was transformed into HT115 (DE3) and expressed according to the method described by Ma et al. [70 (link)]. Control treatments were prepared in the same way but mixed with bacteria transformed with the L4440-GFP vector. Extraction and purification of dsSeNPF and dsGFP were performed by using Zymoclean Gel RNA Recovery Kit (Zymo™, Jiangsu Hongqi Biological Technology Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. After the determination of their integrity, 50 nL (approximately 150 ng) dsRNA was injected into the conjunction between the prothorax and mesothorax of freshly emerging female and male adults of the SE-Sel strain using a Micro 4TM microinjection device (MicroSyringe Pump Controller, World Precision Instruments, Hertfordshire, UK), according to the method described by Ruan et al. [71 (link)]. Their mating ratio and oviposition rates were recorded.
Micro 4tm
Manufactured by World Precision Instruments
Sourced in United Kingdom
The Micro 4TM is a compact and versatile laboratory instrument designed for precise liquid handling. It features four independent channels, allowing for parallel and simultaneous dispensing of small volumes with high accuracy and repeatability.
Automatically generated - may contain errors
2 protocols using micro 4tm
1
Cloning and Silencing of SeNPF Gene in Spodoptera exigua
2
Measuring ApiAT Family Transporter Kinetics in Xenopus Oocytes
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Methods optimised for the study of ApiAT family transporters in X. laevis oocytes have been detailed previously [20] (link). For uptake experiments measuring trans-stimulation, all potential substrates were pre-injected at 25 nl/oocyte using a Micro4 TM micro-syringe and A203XVY nanoliter injector (World Precision Instruments). Oocytes were then incubated on ice for 30 mins prior to uptake experiments to allow for membrane recovery. Stock solutions containing 100 mM of the candidate substrates in ND96 were pre-injected to give a calculated cytosolic concentration of 5 mM, based on an assumed free aqueous volume of 500 nl/oocyte [63, (link)67] . Calculations of cytosolic concentrations from pre-injection should be treated as approximations only, as stage 5 or 6 oocyte diameters vary from 1-1.3 mm and aqueous oocyte volumes range from 368 to > 500 nl [67, 68] (link).
We utilised two different methods for measuring radiolabelled efflux and retention in oocytes. All oocyte uptake and efflux experiments were performed in solutions containing both [
We utilised two different methods for measuring radiolabelled efflux and retention in oocytes. All oocyte uptake and efflux experiments were performed in solutions containing both [
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