How to genotype RNAi bacteria?

Hi guys,

I am very new to worm field. I am asked to pick a skn-1 RNAi from Ahringer library, and also asked to genotype skn-1 RNAi first before I do any experiments with it. I don’t know how to genotype this RNAi. Could anyone tell me how?

I saw Forward and Reverse primer sequences in Ahringer excel file. Can I use the two primers to genotype this skn-1 RNAi?

Thanks!

Kim

Ahringer library clones were generated using TA cloning to put inserts PCR’d from genomic DNA into the L4440 RNAi feeding vector. Assuming you’ve got one or more individual colonies, you have 3 options:

  1. Prep plasmid DNA, and do test digests. This is fast and is the cheapest method, although I suppose it could leave a small chance the plasmid isn’t what you think it is. You can check that the insert is the right size, and can use the primer sequences you found to figure out some sites that should be in the amplified sequence and make sure they’re in your plasmid at the appropriate places.
  2. PCR. Fairly fast (the fastest, if you do it directly from the colony - although only if you obtained the PCR primers ahead of time), and should work, but you need a new primer pair for each different clone. Take the primer sequences you found (or preferably a primer pair internal to that pair from the amplified portion of skn-1), and check that PCR from your colony gives a band of the expected size.
  3. Prep plasmid DNA and sequence it. Not as fast as the other methods, and not cheaper than PCR the first time - but it has a couple of big advantages: you can get sequencing primers that work with the L4440 vector and use them not only with this clone but also with other clones you check in the future (rather than ordering new primers for each clone), and it gives you the most information about just what’s in the plasmid.

Thank you very much! I really appreciate your help! :slight_smile:
Kim

A simple way that I use to verify the clones with little chance they are wrong is to do a PCR from the liquid culture (no prep required) with a T7 primer followed by digest by one or two enzymes (in the PCR mix, split in three: undigested, Restriction enzyme 1, 2. Most work in PCR buffer, neb.com has a table). If all these match the predicted sizes - including the small parts of the multiple cloning site - then it is unlikely to be the wrong clone.