[Automated-sequencing] Re: Automated Sequencing of mRNA/cDNA

Phillip San Miguel pmiguel at purdue.edu
Wed Aug 17 10:50:58 EST 2005


Alex,
	It is possible to sequence RNA templates using reverse transcriptase. 
It has been done. However getting this technique up and running on a 
MegaBACE might be difficult. You would need a primer that binds to a 
unique position on your RNA. Then you would need your ddNTPs to be 
labelled with the correct fluor.
	Also, it is normal now to do cycle-sequencing in standard sequencing 
reactions. You probably will not be able to do this with RNA as a 
template. So you probably will need to use a larger molar quantity of 
your template than you do with your plasmid/PCR templates.
	My advice is to create cDNA and sequence that with your normal kit.
Phillip

Alejandro Gonzalez wrote:
> Hi Phillips.
> Thanks a lot for your info, yes I agree with you.
> By away we may change doing Sanger Sequencing method using SuperScript 
> RnaseH- Reverse Transcriptase (Invitrogen), how I have to do? we need 
> the ddNTPs* and dNTPs and what more?, and we'd like to sequence from the 
> mRNA. Do you have any protocol? can we make by RT-direct Sequencing?
> Our RNA is total RNA from CHO cells.
>  
> Thnks
>  
> Alex G.
> 
>     ----- Original Message -----
>     *From:* Alejandro Gonzalez <mailto:agonzalez at gemabiotech.com>
>     *To:* autoseq at net.bio.net <mailto:autoseq at net.bio.net>
>     *Sent:* Tuesday, August 16, 2005 11:19 AM
>     *Subject:* Automated Sequencing of mRNA/cDNA
> 
>     Hi everybody.
>     I'm working in automating sequencing of the plasmid and PCR
>     templates, using MegaBACE 1000 DNA System Sequencer and the DyEnamic
>     TM ET dye terminator Kit (GE-ex Amersham B.). I'd like to sequence
>     by Sanger method a mRNA template or its cDNA, if anybody know how to
>     do? or any tips to sequence this kind of template?, if anybody could
>     send me a protocol to prepare this sequencing reaction and if I can
>     use same kit (of GE-Amersham) I have been using.
>      
>     Thanks a lot
>     Sincerelly
>      
>     Alex G.
>     Gema Biotech sa



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