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Old 03-04-2015, 03:34 PM   #6
bilyl
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Location: USA

Join Date: Aug 2013
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Quote:
Originally Posted by SDK View Post
Hi All,

I have a question regarding sequencing long-range amplicons affordably, as the title suggests! So, in short I have about 40 long range PCR amplicons from 40 different species (amplicons are ~10kbp). I have no internal sequencing primers. I'm wondering what's my best course of action to get these sequenced. I have a MiSeq in my lab, but the only way I can think to do this is to break up the samples using a Nextera kit which could get expensive. Sanger is out of the question because even if I took the time to develop internal primers, they wouldn't amplify across the 40 species...so I'd have to develop internal primers 40 times! Any other ideas? Please keep in mind that I can't pool the samples as I need to know "who is who".

I'm thinking there has to be a kit out there or a way to do it with restriction enzymes and MIDS?

Thanks for any suggestions!
Why not just randomly fragment the amplicon with fragmentase and then make a library with barcodes corresponding to each one? You could assemble them afterward. You would get around using the Covaris. You could also use Kapa's new HyperPlus kit which has a fragmentation enzyme included. Because these are amplicons you can get away with using a lot less DNA than the recommended amounts.
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