Gen bacteriano y Análisis de Expresión El uso de microarrays

Protocol

Reacción de la transcriptasa inversa A su vez un calentador a 70-80 ° C y 42 ° C. Baños de agua también se puede utilizar en este paso. Si utiliza bloques de calentamiento, poner un poco de agua para que el calor es uniforme. Cebado reacción Tomar 3 g de ARN Ajustar el volumen a 13 ml con agua libre de RNasa Añadir 2,5 l de azar hexamer primers (2mg/ml) Mezclar bien e incubar a 70-80 ° C durante 8 minutos. Después, coloque …

Materials

Material Name	Type	Company	Catalogue Number	Comment
AA-dUTP		Sigma	A0410	5-(3-aminoallyl)-2’deoxyuridine-5’-triphosphate
dNTP		Amersham	27-2035-01	100 mM dNTP Set PCR grade
Random Hexamer primers		Amersham	27-2166-01	3mg/mL
SuperScript III RT		Invitrogen	80800444	200U/μL
CyDye™		Amersham	RPN5661	Post-Labelling Reactive Dye Pack
QIAquick		Qiagen	28104	PCR Purification kit
1M K2HPO4
1M KH2PO4
1M KPO4	Buffer			To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine: 9.5 mL 1M K2HPO4 and 0.5 ml 1M KH2PO4
Phosphate wash buffer	Buffer			For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix: 0.5 ml 1 M KPO4 pH 8.5 + 15.25 mL MilliQ water + 84.25 mL 95% ethanol. Wash buffer will be slightly cloudy. ** IMPORTANT: Phosphate wash buffer should be prepared daily.
Phosphate elution buffer	Buffer			Diluting 1 M KPO4, pH 8.5 to 4 mM with sterile water
Sodium Carbonate Buffer				(Na2CO3): 0.5M, pH 9.0. Dissolve 4.2 g NaHCO3 in 80 mL of sterile water and adjust pH to 9.0 with 10 N NaOH; bring volume up to 100 mL with sterile water. To make a 50 mM solution for the dye coupling reaction dilute 1:10 with water. Note: Carbonate buffer changes composition over time; make it fresh every couple of weeks to a month.