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3' RACE troubles


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#1 Marķa CrS

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Posted 26 August 2014 - 03:41 PM

Hi everybody!

Recently I have started to work with 3' RACE using invitrogen kit for both ends (5' & 3'). I am just interested in the 3' end.  At the beginning I used a primers  that amplifying a known  fragment of  my gene  (with an amplicon of 600 bp). These primers were used to corroborate the presence of the transcript of interest were present in the tissue. If I synthesize cDNA using  oligo dT from superscript III and then perform a pcr to amplify a house keeping gene and the known fragment of my gene of interest,  both of them are amplified. In contrast if I used oligo dT of the kit RACE invitrogen to synthesize cDNA using the same source of RNA, and then performing the PCR to try to  obtain the unknown 3' fragment using as controls pcr reactions for amplifying the housekeeping gene and the known fragment of my gene of interest I can't amplify the known sequence at least. Housekeeping gene is amplified and controls of the kit race also are ok. I have no idea what is the problem?.... Any idea to solve this mystery??, has somebody had the same problem??

Thank you in advance for your help!!



#2 Ant_Ony

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Posted 27 August 2014 - 12:32 PM

Hi Maria, 

I also started recently to work with 3'RACE, but it´s not working very well for me, too.

To be honest, I did not get exactly what your problem is.

Did I get it right, that it´s working well, unless you use the oligo dT of the invitrogen RACE kit?

This would be interesting for me, as I am also using the invitrogen kit.

I am not sure if I can help you, as I don´t have much experience with RACE...

Best,

Ant_Ony



#3 sezerf

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Posted 20 October 2014 - 02:36 AM

Hi Maria;

Your problem is more likely to be related with PCR reactions. Are you doing the nested amplification? If you dont get any product from nested amplification allthough you tested your RNA you should check and maybe order new primers.

 

Scotto-Lavino E1, Du G, Frohman MA. 3' end cDNA amplification using classic RACE.Nat Protoc. 2006;1(6):2742-5.  < I Have used this protocol and commonly used PCR cycle conditions differ from classic PCR protocols. (Like an extended first cycle (40 min extension). If ı understand right you use same conditions with your controls. If so maybe you need to try other PCR protocols. 
 
Best
Fatih





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