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, Protocoles de transfection Pour obtenir des cellules exprimant un plasmide qui code pour un uorophore simple ou un biosenseur, nous décongelons les bactéries contenant ce plasmide et nous en faisons une culture. Puis nous purions l'ADN plasmidique selon les étapes du protocole "Xtra Midi Finalizers , high copy" : 1. Centrifuger dans un falcon de 50mL à 5000 rpm pendant 15 min à 4 ? C

, Resuspendre avec le milieu RES 8 ml. Aspirer refouler pour bien diluer le culot

, Ajouter le milieu LYS 8 ml, remuer en fermant 5 fois. Incuber pendant 5 min à température ambiante

, Ajouter le milieu NEU 8 ml, agiter doucement en renversant le tube jusqu'à ce que le milieu devienne blanc

, Centrifuger à 6500 g pendant 10 min

, Placer la colonne avec son ltre dans falcon de 50 ml et les laver avec le milieu EQU 12 ml en le versant sur les bords du ltre

. Verser-le-lysat-contenant-l'adn-dans-le-ltre,

. Enlever-le-ltre,

, Mettre la colonne sur un tube NEUF (ADN récupéré ici)

, Verser le milieu ELU 8 ml

, Enlever la colonne et ajouter l'isopropanol 3,5 ml

, Nous pouvons ensuite réaliser la transfection à proprement parler : 2. Dégazer 30 min sous cloche à vide 3. Nettoyer la galette en silicium avec de l'isopropanol et de l'air propre 4. Verser le mélange de PDMS sur le wafer dans une boîte de pétri 5

, Décoller le PDMS cuit du wafer et le découper avec un cutter Impression des protéines d'adhérence : 1. Préparer une solution de 97 µl PBS + 2 µl Fibronectine + 1 µl Fibrinogène 2. Déposer cette solution sur le tampon et incuber 5 min

, Aspirer la solution et bien sécher le tampon à l'argon propre 4. Poser le tampon sur le PDMS mou, appuyer légèrement 5

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