Réaction de la transcriptase Tourner bloc chauffant à 70-80 ° C et 42 ° C. Bains d'eau peut également être utilisé dans cette étape. Si l'aide de blocs de chauffage, mettre un peu d'eau afin que la chaleur est uniforme. La réaction d'amorçage Prenez 3 pg d'ARN Réglez le volume à 13 ul avec de l'eau RNase Ajouter 2,5 pi de hasard hexamère amorces (2mg/mL) Bien mélanger et incuber à 70-80 ° C pe…
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.