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PCR product sequencing

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3 replies to this topic

#1 yyc



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Posted 23 November 2013 - 01:14 PM



I would like to know if it is possible to sequence direclty a PCR product without first cloning the fragment.


Thank you.

#2 hobglobin


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Posted 23 November 2013 - 01:24 PM

yes it's possible, but you have to be aware of some topics such as that is has to be really only one product (and not a mix of two or more similar long fragments, therefore checking it on a gel is recommended, or even cutting out from gel to have them separated), cleaning up the product is essentially too, to get rid of nucleotides and other unwanted stuff.

Search for this topic here, there are several threads with useful hints.

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#3 pcrman



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Posted 23 November 2013 - 02:14 PM

Sequencing PCR products without cloning is a common practice. If your product is a single band (by running a small aliquot), you can sequence it after purification without gel separation, otherwise, you have to gel separate it, cut out the band of interest, and recover the DNA from the gel slices. 

#4 El Crazy Xabi

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Posted 05 December 2013 - 10:11 PM

If you amplify a single copy-gene (or multi-copy, conserved in sequence) comming from pure culture or individual is more than common. I'd say is the normal procedure for example for sequencing ribosomal SSU, ITS or LSU. I do it for identification of isolates and I never did any cloning nor gel geparation, just DNA cleanup (that's a must). Depend on what do you want it for.

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