How can I analysis the transcript after T7 promoter? - [HELP] (Aug/14/2006 )

I have cloned a sequence (about 50bp length) into the MCS of a plasmid.
There is a T7 promoter before the MCS.
Now I am to make my sequence transcribed to a short piece of RNA,
However, I have no idea about how the product RNA will be.

It said that there are certain software to analyze it.
Would you please tell me what kind of software it is? and where can I get the software?
Thank you very much!

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I'm a little confused.

So you've cloned a 50 bp region with unknown sequence and you want to deduce the sequence.

Is that right? Or have I got the wrong end of the stick?

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One way to do it is to use a form of Sanger Sequencing. Using deoxyribonucleotides. Here is a link:

http://www.pnas.org/cgi/content/abstract/75/11/5334

You can download the pdf for free. Notice the paper is from 1978.

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Thanks for the reply.

I do know the sequence that has been cloned into the vector, however, I do not know the start and the end point of transcription of the vector.

It is pcDNA 3.1(invitrogen), the vector, with a T7 promoter in front of the MCS, and a SV40 adding after the MCS.

I have asked invitrogen's tech support, they told me to analyze the transcript by some software, however, I have no idea what kind of software can do it.

So, do you know such software?
Thanks a lot~

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I guess they mean sequencing software.

First you have to set up an in vitro transcription system. Then you need to sequence the transcript and analyse the results. This can be done using software or by eye if it has worked really well.

The quickest and easiest way is to send the plasmid to a professional sequencing unit. Do you work in a university? They're almost certain to have their own facility. It can be quite affordable and very swift. Ask about. This will save you all the bother.

You may find you need to clone your sequence into a different plasmid depending on the primer sequences the facility uses.

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Thank you very much for you help:)

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for folding predictions you can use mfold which is free and you just have to put the sequence of interest and calculation is done quick

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so, we cannot predict the transcript before actual transcription?

anguished~

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Not unless you know the sequence of the DNA template

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I know the sequence,

How can I predict the transcript?

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Um,

A --> U
T --> A
G --> C
C --> G

Am I missing something?

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