[Automated-sequencing] Re: Automated Sequencing of mRNA/cDNA
Phillip San Miguel
pmiguel at purdue.edu
Wed Aug 17 10:50:58 EST 2005
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.
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.
> 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
> Alex G.
> Gema Biotech sa
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