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Electrophoretic Mobility Shift Assay Forum EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


Cross-linking T7 RNAP to T7 genomic DNA

Cross-linking T7 RNAP to T7 genomic DNA - Electrophoretic Mobility Shift Assay Forum

Cross-linking T7 RNAP to T7 genomic DNA - EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


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Old 01-20-2010, 09:11 AM
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Default Cross-linking T7 RNAP to T7 genomic DNA



Hi...

I would like to cross-link T7 RNA Polymerase with T7 genomic DNA in-vitro. Both the RNAP and genomic DNA were purchased commercially. The interaction between the RNAP and T7 DNA are very weak. I would like to fix the proteins to the DNA to facilitate my experiments. Is there any method I can follow to do this? I have heard of formaldehyde cross-linking and UV cross-linking methods but I would like to have some specific protocol to try out.

Have a good day!!!
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Old 01-21-2010, 02:20 AM
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Default Re: Cross-linking T7 RNAP to T7 genomic DNA

Hi, no need to crosslink with formaldehyde UV-crosslinking is usually enough.

I would focus on buffer conditions, HEPES vs X Buffer; pH; Salt conditions; Mg conditions etc and optimize your binding first.

Cheers
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Old 01-25-2010, 11:10 AM
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Default Re: Cross-linking T7 RNAP to T7 genomic DNA

Thanks... What is the best method to check the results for the DNA-protein binding? Will gel electrophoresis give good results to make sure whether some binding reaction took place? I am concerned because the binding between T7 DNA and T7 RNAP is very weak.
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