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Old 11-13-2014, 05:33 PM   #4
Location: Perth

Join Date: Sep 2012
Posts: 55

Hi esherman,

A few options/opinions to offer on this.
1) If you do decide to go 2-step you risk contamination. These workflows are really really hard to keep clean. Personally I 'hate' this approach but it does kind of depend on what you are using the data for.
2) Do you need to do paired end sequencing? - we are getting good single direction sequencing out to 250+ bases (on v2 300 nano kits), so if your amplicon is shorter than that you could consider it (i.e you only need a sequencing primer at one end).
3) Use a custom sequencing primer (e.g one with LNA's) to shorten the the total length of the primer you need. Search some of the seq-answer threads on this for a few hints or PM me.
4) a combination of points 2+3 above is what we (mostly) use to keep primer sizes down which helps cost and PCR efficiency.

best of luck with your workflows, Cheers Mike

Last edited by bunce; 11-13-2014 at 06:16 PM.
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