How can I extend my sequencing results?

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sahart
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How can I extend my sequencing results?

Post by sahart » Mon Jan 17, 2011 12:24 pm

Hi all,

I have sequenced around 400 bp of a cDNA, I am wondering how can i extend my sequencing result to whole the cDNA, kindly noted that I have no information about other part of this mRNA.

Thank you, :)

Sahar

magicsiew
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Post by magicsiew » Mon Jan 17, 2011 4:04 pm

carried out RACE-PCR

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canalon
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Post by canalon » Mon Jan 17, 2011 11:22 pm

Sequence walking: Design a new primer at the end of the one you have.
Patrick

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sahart
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Post by sahart » Tue Jan 18, 2011 4:09 am

Thanks all

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Re:

Post by sahart » Tue Jan 18, 2011 4:11 am

canalon wrote:Sequence walking: Design a new primer at the end of the one you have.


How much should I add cDNA as template?

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canalon
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Post by canalon » Tue Jan 18, 2011 4:22 am

Exact same reaction, new sequencing primer, you move a few hundred bp forward until you reach the end. Long but that is what used to be done in the olden days before shotgun sequencing. And if you have access to a good sequencing facility, where read length of above 700bp are the norm that makes things even faster.
Patrick

Science has proof without any certainty. Creationists have certainty without
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JackBean
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Post by JackBean » Tue Jan 18, 2011 6:39 am

if we talk about one read, there is not much what you can do as a customer. Just provide enough high-quality DNA and primers which anneal fine.
http://www.biolib.cz/en/main/

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Re:

Post by magicsiew » Tue Jan 18, 2011 2:25 pm

canalon wrote:Sequence walking: Design a new primer at the end of the one you have.


Hi, if I need to elongate the 5' direction?

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JackBean
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Post by JackBean » Tue Jan 18, 2011 3:49 pm

just design reverse complement primer ;)

But I think, that chromosome walking is basically the same as RACE-PCR ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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