We are embarking in a medium-large scale project. We want to clone cDNAs (full size when < 10kb) of 50-100 genes. Next, we want to use these amplicons for targeted re-sequencing. 2 questions:
1- best strategy for cloning all these targets (TOPO-cloning?)
2- Best kit to perform targeted re-sequencing (Nextera XT DNA Sample Preparation Kit?)
I've had problems of insert stability when using TOPO-cloning..... With inserts that size (10 kb) I'm not sure that cloning be the best option. Manipulating such a long clones could be a nightmare
Hi Estanis, most of the insert will be less than 5 kb. One of them is 17kb and I agree, it's a nightmare. Maybe we will modify the strategy in 5kb subclones fragments.
Hi Luca, in my experience 5 kb is the frontier among a difficult cloning and a never-ending story. If you already know the sequence try amplifying 3-4 kb overlaping fragments with an error-free polymerase. It's a lot of work, but it can be handled.
Another question is about the reverse transcriptase. Is there anyone that copy up to 17 kb?
Good luck!
You can order a chip with thousands of oligos encompassing your genes of interest, and avoid any of the cloning difficulties. Other alternatives exist as well.
A good review is this one:
Target-enrichment strategies for next-generation sequencing.
Mamanova L, Coffey AJ, Scott CE, Kozarewa I, Turner EH, Kumar A, Howard E, Shendure J, Turner DJ.
Nat Methods. 2010 Feb;7(2):111-8. doi: 10.1038/nmeth.1419. Review.
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