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I want to clone ~100 different coding sequences from human or mouse into an expression vector to analyze phenotypes in tissue culture. I can do all of the steps, but I'm not sure what I should use as the template for my reactions. Ideally, I would purchase a pool of full length cDNAs from a bunch of organs, and amplify from that. Less ideally, I can generate cDNAs myself, from multiple organs and pool them. Does anyone have suggestions on a template source?

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  • $\begingroup$ If you plan to take one isoform for each one of the genes, then I see no point in amplifying them pools of tissues. $\endgroup$ – alephreish Sep 24 '14 at 20:31
  • $\begingroup$ The goal is to do a miniscreen for proteins of annotated with a specific function (there are about 100 genes). The idea is to automate the task as much as possible, without spending too much time choosing isoforms or fusion points. I just don't have experience doing this, so I'm not sure if such libraries exist, or if I essentially need to start from scratch from cDNAs and gene specific primers. I figures that by pooling cDNAs I would most likely amplify the most abundant isoform. $\endgroup$ – Mark B Sep 25 '14 at 4:35
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  • Verify where these transcripts are generally present (this should not be difficult for common organisms)
  • If they are all expressed in a certain tissue use cDNA from that tissue
  • If not then pool the cDNAs from different tissues such that all genes can be amplified
  • cDNA libraries (including whole organism) are also available and you may obtain them from the nearest repository (I guess NCBI also provides).

Some other things you can do:

  • Ask the primer synthesis company to provide primer sets it in a 96well format.

  • DNA synthesis is also a decent option (it takes something like $0.5 per base pair), but that can be expensive if your cDNAs are huge.

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