बैक्टीरियल जीन एक्सप्रेशन प्रोटीन का उपयोग विश्लेषण

Protocol

रिवर्स ट्रांसक्रिपटेस रिएक्शन 70-80 हीटिंग ब्लॉक मुड़ें डिग्री सेल्सियस और 42 डिग्री सेल्सियस जल स्नान भी इस चरण में इस्तेमाल किया जा सकता है. यदि हीटिंग ब्लॉकों का उपयोग, कुछ पानी इतना डाल दिया…

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.