cDNA clone

Model Organism: Worms Methods & Reagents
1

Hi
Is anyone aware of how and where I can obtain cDNA clone for my gene of interest to perform PCR amplification since I am finding this to be troublesome with standard cDNA prepared from wild type worm?Is there any source for C. elegans cDNA clones?
Thanks

2

I have often gotten cDNAs from Thermo.
http://www.thermoscientificbio.com/gene-expression-cdnas-orfs/non-mammalian-cdnas-and-orfs/c-elegans

The C. elegans ORFs, RNAi clones, and Transcription Factors section all have cDNAs. Sequence verify any clone, as I have had incorrect clones sent; I think that there are errors in the library. Still, if you don’t want to clone out the cDNA yourself this is a good place to look.

best,

Jordan

3

Hi,

just as a matter of interest, what exactly is troublesome with your PCR when using the self-prepared cDNA of your GOI?

Regards

Steve

4

Hi Steve
The problem i have amplifying from my own cDNA stock is that I either never get the band of expected size (2.6kb) or see very faint band close to 500bp. I have tried using different dilutions but it hasnt worked. If you have other ideas, that would be helpful.
Thanks

5

Hi,

there are a number possibilities for your PCR results…and you could always exclude some of the more common ones by running some ‘control’ PCR reactions.

I’m assuming that you made your cDNA from extracted RNA and then PCR’d this CDNA template with your GOI primers?

General reasons why you might be seeing a band of the wrong size include (and some are more far fetched than others I know…);

  1. incorrect primer design (are you sure that the primers should pick up the full-length GOI
    cDNA?).
  2. Is it possible that you are picking up a splicing product?
  3. contamination.
  4. mispriming of primer, really a variant on 1. with reaction kinetics mixed in…
  5. incorrect annealing temperature.

We find you need relatively little from the prepared cDNA to achieve decent PCR results if all other aspects of the reaction are OK.

Is it possible for you to try using a primer set that you (or someone else in your lab) know works and do the PCR on your cDNA with these primers. This at least excludes the general reaction conditions and the standard of ther cDNA as possible confounding factors?

Regards

Steve

6

I agree with Steve, you should ask someone for some primers that have been used to successfully amplify a cDNA and test it on your cDNA prep. If you think your primers are bad, try using them on just genomic DNA and see if you are able to amplify anything (which you should).