Hello everyone! I am using the Ambion Firstchoice RLM-RACE kit to get both extremes of two mRNA from a Diatom. For the 3'-RACE i did't experience any complication, but so far I am still not able to get the 5'-end in any of the two targets. I have tried with different amounts of input RNA and different samples, always following the manufacturer's instructions, without success.
The expression of the mRNA was checked by RTPCR with internal primers. Even after finishing 5' protocol I am able to amplify both genes (so RNA/cDNA is not being degraded)...but when I do the nested PCR using the combination of the 5'adapter primer and my specific primer (outer and inner) I just don't get anything. I also increased extension time during PCR to 1.5 min (the expected size of the fragments is less than 600bp) to be sure that the partial fragment will be amplified, but nothing.
I am afraid that the adapter is not getting attached properly to the 5'-end...does anyone know why this could happen? Is it possible to check after all 5'-RACE steps that they actually worked well?...Any advice you can give me?
Thanks a lot!!
Earlier during 5' RACE me too faced such situation ... but i have to leave that project as i was selected for Ph.D
their could be one more possibility , if you have checked the adapter and your primers make sure that they are not making any dimers..
nice question.
All the best
I found clontech SMART RACE kit is the best. I used the old version kit for many of my experiments. Now they have the new version: Smarter RACE kit, I think they are the same. Try to use a touchdown PCR prog. to ampify your products. Within 2-3 rounds PCR, you should be able to obtain the full-length of your gene.
Hi,
I have no experience with abovementioned kit, however I guess, the terminal transferase step is a crucial one. Check, that your enzyme does work (eg by analyzing of a single product on a gel), or run several variable conditions for TdT. Also make sure, your TdT buffer is compatible with downstream application (sometimes contains CoCl2 and the product needs to be purified). Good luck
Thanks for your answers.
-Parakesh: I have some product of primer dimer, but I don't think that is the reason I don' have a product of my GOI...I tried with another set of primer that don't produce any secondary structure (theoretically) and also didn't work.
-Kai: Maybe if I try with another kit I won't have this problem. unfourtunately, they are very expensive and comes with very few reactions.
-Martin: This kt uses an adaptor ligation step and no terminal tranferase protocol.
I repeated the nested PCRs after 5' RACE reaction increasing the amount of imput cDNA and, finally, I got a product that I am quite sure that could be one of the genes I am studying, according by its size (of course the only way to be sure is sequencing), but for the other one it doesn't matter what I try, there is no product....
Do you think that secondary structures of the mRNA of my gene can interfere with the first steps of the reaction (decapping and adapter ligation)?
cheers
-Martin: You are right.TdT is very critical. But there is no need to use TdT in Clontech kit.
- Erik: To save money, you might order the primers yourself according to the user manual. The other reagents needed for the experiment are very common. For the reverse transciptase and Taq polymerase, either you can order the same from the kits. Or if you have your own reverse transciptase from you the lab, you can also use it. i.e. Superscript Reverse transcriptase from Invitrogen is fine. For Taq, use the one can amplify for long target, such as Expand™ Long Template PCR System , Expand™ High Fidelity PCR System and KAPA Long Range system.
Pls. find the attached gel picture of my 5' and 5'- 3' of 2 separate expts. and Touchdown PCR cycle.
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