Hello Emiliano,

Thanks for the advice. Of course the northern!

Jonathan

Hi Jonathan,

I would recomend you also to try the adaptor approach instead of the oligo d(T), it seems to me that is likely that you are getting amplification from that small A strech in the midle of your sequence.

Good luck!

This is great...I have also been having some trouble characterizing 3'UTR polymorphism. Emiliano, do you have a publication reference or the name of a kit you use to ligate your DNA adaptor to the 3'end of RNA with the T4RNAligase2 ?

Hi Jonathan, I think Emiliano's answer is great. I would like to add one simple test: You can design two nest froward PCR primer, one is from upstream of your putative product, second one is from the downstream. (you know the distance between two nest primers). The reverse primer design, follow Emiliano's sugestion. The nest PCR reaction should give you two product with expected size difference. This way will tell you whether the truncated product is real or not.

thank Emiliano's great answer. I am thinking maybe both of them could be correct, there could be some non-functional or error copies, together with the correct ones.

This is another good protocol.

Article 3' end cDNA amplification using classic RACE

Thank for Emiliano's answer. This will help a lot in my experiments.

Ambion has a very nice kit for that: FirstChoice® RLM-RACE Kit.

https://products.invitrogen.com/ivgn/product/AM1700

You remove the 5´-CAP, ligate an oligo to the remaining 5´-phosophate and perform RACE PCRs. Works very well in our hands.

Hello, i'm looking for a RACE PCR KIT that enables Products amplification and sequencing without the cloning step do you know if such a method is available by any producer ? thanks for the help

I found the: 5’/3’ RACE Kit, 2nd Generation from Roche that allows RACE PCR products sequencing without cloning, has anyone tried it any thoughts? thanks