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3' and 5' RACE PCR

3' and 5' RACE PCR - RNA Forum

3' and 5' RACE PCR - RNA Forum. Discuss RNA including mRNA structure and function, rRNA, tRNAs, and the biochemistry of other RNA molecules.


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Old 04-16-2010, 06:28 AM
Pipette Filler
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Default Re: 3' and 5' RACE PCR

yes, u need to run it before any attempt of cDNA synthesis. if RNA is not good or absence, of course your downstream process will not successful at all. my suggestion is, try to run your RNA sample in 1% of AGE at 100volt for 30 minutes. if your RNA is in good condition, you should get double band (or more). that double band represent 18s and 28s (if im not mistaken) RNA which is abundant in any living cell.

your 2nd question: yes, im using the same reverse primer. because my primer were designed with modified nucleotides. thus it is suitable for PCR. but your kit, if it says do not use the same primer, your should use its nested primer. read your manual properly.

i just wanna share one experience with you, my colleague study on goat. she retrieved sample from goat's saliva. NONE RNA/DNA were able to extract from those samples. so, you should reconsidering on how to get RNA. the best is cow's blood of cow's meat. choose which ever suit for your case

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Old 12-01-2010, 06:19 AM
Pipette Filler
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Default Re: 3' and 5' RACE PCR

Hi, I'm also facing the problem of getting any positive result for my 5'- and 3'-RACE using SMARTer RACE cDNA amplification Kit (Clontech).
I can get multiple band for my 5'-RACE for the first time i do 5'-RACE with this kit, which the multiple bands may due to my gene is a family genes. Unfortunately, i cannot get anything after using newly synthesized cDNA. I had synthesized new cDNA for twice, but all failed in getting any band in my RACE.
I had check my cDNA using other primer pairs, the targeted band was observed. So this mean my cDNA still ok. I had also re-designed my GSP, and i still get nothing.
Can suggest any problem that may cause this?
Really need some help to solve my problem cos i have stuck here for half year already.
Thanks ya =p
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