Bacteriële Gene Expression analyse met behulp van Microarrays

Protocol

Reverse transcriptase reactie

Draai verwarming blok naar 70-80 ° C en 42 ° C. Waterbaden kan ook gebruikt worden in deze stap. Bij gebruik van verwarmings-blokken, dus zet wat water dat de warmte uniform is.

1. Priming reactie
   1. Neem 3 pg RNA
   2. Volume aan te passen tot en met 13 ul met RNase vrij water
   3. Voeg 2,5 ul van willekeurige hexameer primers (2mg/mL)
2. Meng goed en incuberen bij 70-80 ° C gedurende 8 minuten. Dan, plaats op ijs gedurende 5 minuten.
3. Bereid RT cocktail (14,5 ul / rxn):
   
   

   Bestanddeel

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AA-dUTP		Sigma-Aldrich	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
1 M K2HPO4
1 M KH2PO4
1 M 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.