Pyrosequencing of the midgut of the Leptinotarsa decemlineata larvae
Midgut from multiple individuals from a laboratory stock of Leptinotarsa decemlineata larvae. First strand cDNA was synthesised using the SMART PCR cDNA Library Construction Kit (Clontech). Double stranded cDNA (ds-cDNA) was synthesised and normalised following the Trimmer Kit for cDNA normalization (Evrogen) guidelines. The optimal condition for ds-cDNA synthesis for each single stranded cDNA template was empirically determined: reaction mixes were subjected to a recommended range of thermocycle numbers and their products checked by electrophoresis (1 % agarose gel). The optimal cycle number was defined as maximum cycle number without signs of overcycling. Ds-cDNA was then purified and concentrated using the DNA Clean and Concentrator kit (Zymogen).