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T7 RNA polymerase transcription and translation.


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#1 Niraj

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Posted 08 June 2010 - 11:23 AM

Hi All,
I am having an unique problem. I have my gene of interest (GOI) cloned in pcDNA3. Then we mutated the first ATG from my GOI. Now when I tired to do in vitro transcription translation (Promega) using the first ATG mutated gene there is no radioactivity. On the other hand my wild type GOI is showing nice product. So, my question is what is the maximum number of nucleotides you can have between the T7 promoter and first ATG? Please support your reply with the reference article if you can.
Thanks a lot.
Regards,
Niraj

#2 pDNA

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Posted 08 June 2010 - 12:00 PM

in vivo the spacing between RBS and ATG should not exceed 13 nucleotides ...for more information see this review.

I would suggest it is very much the same for in vitro applications.

Regards,
p

Edited by pDNA, 08 June 2010 - 12:00 PM.


#3 Niraj

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Posted 09 June 2010 - 02:06 PM

Thanks a lot pDNA.

in vivo the spacing between RBS and ATG should not exceed 13 nucleotides ...for more information see this review.

I would suggest it is very much the same for in vitro applications.

Regards,
p






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