बैक्टीरियल जीन एक्सप्रेशन प्रोटीन का उपयोग विश्लेषण

Published: May 28, 2007
doi:

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.

References

  1. Hasseman, J. TIGR Aminoallyl Labeling of RNA for. Microarrays & TIGR Microarray Labeled Probe Hybridization. , .
  2. Gilbert, , et al. TIGR Microbial RNA Aminoallyl Labeling for Microarrays & Hybridization of probed labels. , .
  3. Hedge, , et al. A concise guide to cDNA Microarray analysis-II. , .

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Cite This Article
Beyhan, S., Yildiz, F. Bacterial Gene Expression Analysis Using Microarrays. J. Vis. Exp. (4), e206, doi:10.3791/206 (2007).

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