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Newbie to Sequencing Question


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#1 MaryNelson

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Posted 29 August 2010 - 05:54 PM

I've performed Site Directed mutagenesis on a 2500+bp stretch, and need to sequence it to make sure there are no unwanted mutations.

I've got several different primers for the sequence to choose from available, but I'm not sure how many it's best to use, or where along the sequence to aim.

Any help/advice? Thanks!

#2 Clare

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Posted 30 August 2010 - 12:40 AM

Hello :)

If you want to sequence your region of interest to see if there are any mutations, you want your primers to cover the whole area (and have both forward and reverse primers to sequence both strands). I usually use primers positioned ~ every 700bp or so (but it depends on yoru read length).
Hope this helps!
Clare

I've performed Site Directed mutagenesis on a 2500+bp stretch, and need to sequence it to make sure there are no unwanted mutations.

I've got several different primers for the sequence to choose from available, but I'm not sure how many it's best to use, or where along the sequence to aim.

Any help/advice? Thanks!






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