RNAi Clone Sequence Verification


I would appreciate some advice. I sequenced an RNAi clone to verify my gene of interest from the library. BLASTing shows the top hit for my gene. However, the sequence I got back aligned to only half of the gene. This is because (for logistical reasons) I only used the forward (M13) primer. Is it safe to assume that the clone contains the correct insert? Or has the RNAi library been known to have inserts that are not full length? And even if that is the case, what are the odds that dsRNA will not be knocked down if only half the gene is present?

In our experience, it is rare for sjj and sjj2 clones to be truncated. mv clones will correspond to a single transcript and so if there are alternative exons between 5’ and 3’ primers, the insert may be substantially shorter than the longest predicted transcript.

As far as targets, this tool we made might help: http://bioinformatics.lif.univ-mrs.fr/RNAiMap
[See http://www.g3journal.org/content/4/11/2137]

Half of the gene or half of the expected insert? Few of the Ahringer clones cover the entire gene. But you can find the primers used in Wormbase, under “reagents”