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-   -   3' and 5' RACE PCR (http://www.molecularstation.com/forum/rna-forum/26277-3-5-race-pcr.html)

Noumy 10-29-2008 12:05 PM

3' and 5' RACE PCR
 
Hi guys,

Would anyone of you have an interesting website or scheme for the 3' and 5' RACE PCR. I am desperately looking for one, but don't find anything.

Thanks a lot in advance!



Noémie, new on this forum :)

zulcyq 10-29-2008 03:55 PM

Re: 3' and 5' RACE PCR
 
hi Noémie,

try this, kinda helpfull. [Only registered and activated users can see links. Click Here To Register...]

besides, im doin RACE now, n i would suggest you to use FirstChoice RLM-RACE kit (Ambion)

and last advise, why dont you look for the original RACE paper by Frohman et al 1988 which help you to understand better RACE concept so you can proceed to experimental level.im done it during my first time

try out!

regards
CyQ

Noumy 10-30-2008 07:25 AM

Re: 3' and 5' RACE PCR
 
Hi CyQ,

Many thanks for your rapid answer! I won't do such PCR before few months, but I noted your advises =)

Cheers from France,


Noémie

begoroibas 02-25-2010 06:06 AM

Re: 3' and 5' RACE PCR
 
Hi CyQ,

I was trying to find the article that you mention before but I din't find it.

Do you mind to send me the link?

Thanks for you help!!!!

zulcyq 03-01-2010 03:19 AM

Re: 3' and 5' RACE PCR
 
Hi begoroibas,

this is title for Frohman journal

Rapid production of full-length cDNAs from rare transcripts:
Amplification using a single gene-specific oligonucleotide primer

from PNAS. you should find it. sorry cant give you the link. ive done 3' race and 5'race manually using this protocol. some modification was applied. the equipment, specific primer and DNA taq polymerase and PCR machine. ive done it to my 1st gene. now working on 2nd - 4th gene. so, i hope that you can do it also.

cheers!! :)

begoroibas 03-24-2010 04:23 AM

Re: 3' and 5' RACE PCR
 
Hi CyQ:

I am doing now RACE-PCR with invitrogen kit.I didn't get any results yet.

I think that mi GSP1 primer desing is not good,could you help me?

How did un desing your primers?Thanks!!!

zulcyq 03-24-2010 06:06 AM

Re: 3' and 5' RACE PCR
 
Quote:

Originally Posted by begoroibas (Post 419483)
Hi CyQ:

I am doing now RACE-PCR with invitrogen kit.I didn't get any results yet.

I think that mi GSP1 primer desing is not good,could you help me?

How did un desing your primers?Thanks!!!



Hi. To be honest, ive never use any of RACE kit. ive improvise alot of the technique. well, if you ask me how im designing my GSP1, use web-based tools. currently im doing Primer3 at ([Only registered and activated users can see links. Click Here To Register...]). you can try them. might give you a better result. one thing, try to do temperature gradient on primer annealing step. it might give significant result on different temparature.

cheers!

begoroibas 04-13-2010 06:28 AM

Re: 3' and 5' RACE PCR
 
Hi again!

I am completely desperate.My boss spent a lot of money in the RACE kit and I can't make it work.
I am going to order new primers,and i hope it does.I am wondering about your protocol without the kit.
How do you perform the RACE PCR?

You start from RNA,using SuperscriptII you got your cDNA ,and after that?

I think that you are the only one in this world who can help me!THANKS!!

Bego

zulcyq 04-14-2010 05:21 AM

Re: 3' and 5' RACE PCR
 
hai bego.
for me, u should get it. i mean, get the best result. most paper on getting full length of any gene used kit instead of manual technique like i do. more easier and the fastest way to get it. nvm, i will share with you on how to do it.

well, my RNA is from high polysaccharides plant. ive extracted them using modified technique from Gasic et al who was succesfully extract RNA from apple. then, with a very good RNA band then i proceed to DNase treatment and cDNA synthesis. primer used in cDNA synthesis should be provided in your kit right? may i know what kit you are using now? maybe i can look for its manual and its primer. besides, currently im using totally different primer which originally design for DDRTPCR, not for RACEPCR. nevertheless for me, any anchor primer works. as long as you know on how to manipulating them. after that then i'll do PCR using the same primer to do cDNA. this primer contain modified nucleotides to make it stable. the forward primer that ive used design from web-based program named Primer3. you may design them using various type of program available such as NCBI o etc...

for your case, i would say, maybe your RNA itself is not good enough. try to extract more better RNA.

cheers

begoroibas 04-15-2010 05:23 AM

Re: 3' and 5' RACE PCR
 
Hi CyQ:

Thank you very much for your answers.I really appreciated it.

I am using the INVITROGENE RACE PCR KIT.With this kit you have to desing 2 primers,GPS1 and GSP2.
The first one is for cDNA synthesis and the second one is kind pf nested pcr.

I need to isolate the promoter of the Oct4 gene in cow.

I think that one of the problems is my RNA condition.Before starting to perform cDNA synthesis,should I run a gel with the RNA sample?
And another question,do you use the same reverse primer for cDNA synthesis and PCR?

Thank you very much,you are being so kind!


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