SEQanswers - View Single Post - 'Assembly PCR' for homemade amplicon library prep

bunce · 11-13-2014, 05:33 PM

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

11-13-2014, 05:33 PM	#4
bunce Member 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.