Where to buy RNAi? How to make RNAi?

Model Organism: Worms Methods & Reagents
1

Hello,

I have been buying my RNAi feeding clones from Thermo Scientific Open Biosystems. Is this a good source? From where do you guys get your RNAi E. coli?

Also, sometimes I want to do RNAi against a certain gene, and the E. coli is not available from Thermo. How can I make my own RNAi clone? To my understanding, it should be quite simple, just inserting the dsDNA of choice into the MCS of pL4440. But does anyone have a protocol??

Thanks for the help,
auphorie

2

Hi Auphorie,

You are correct in that you can clone your gene of interest into L4440 (pPD129.36), which can be purchased from Addgene if you do not already have it. Or you should be able to obtain it from any local worm lab. However, the OpenBioSystems clones are only from the Vidal ORFeome RNAi library. The Ahringer library clones are available from SourceBioSciences. Perhaps your clone of interest is not in the ORFeome set but is in the Ahringer set.

If you choose to make your own, you will also need HT115 bacteria, which can be obtained from the CGC (https://dbw6.msi.umn.edu/cgcdb/strain.php?id=8854).

Good luck!

Steve

3

Hi Steve,

Yes, I have looked at SourceBioSciences. It costs $926 USD just to buy one clone, which is way too expensive. Are there any other options out there?

Thanks!

4

Does every C. elegans lab use E. coli strain HT115 for feeding RNAi? Wouldn’t a strain with a repressor work better? HT115 seems a little leaky, in some cases inducing depletion even without IPTG.

5

Making your own RNAi clone is easy.

  1. Select a restriction site from the MCS of L4440.
  2. Design PCR primers for your gene(s) of interest; add three ‘A’s plus the restriction site at the 5’ end of each primer.
  3. Amplify genomic or cDNA with primers.
  4. Digest L4440 and PCR product with restriction enzyme; treat L4440 with phosphatase to prevent recircularization.
  5. Clean up digests; ligate together; transform into bacteria.
  6. Screen clones by PCR; recover DNA from positives; confirm by restriction digest.
  7. Transform into HT115.

Also, it costs a lot less than $926 ;).

Harold

6

For people with PCR handicaps or wanting a full-length cDNA clone (which can sometimes be more effective) you can order Kohara cDNA clones and cut and paste inserts into L4440.

7

My experience is that you can even skip steps 5 and 6 and transform/screen clones into HT115 directly… the efficiency is worse than using a Top10/DH5alpha kind of intermediate, but I’ve rarely had no positive colonies from just using HT115

Either way, though, follow this protocol and you should be fine!