I have 200 bp known fragment. for RACE PCR. I am using Clontech kit. I tried all the possible primers with UPM (with kit), but never got the desired band. I synthesized new RACE cDNA but still the problem persists. The degenerate primers work well to get partial fragment, but GSPs dont. I can't use one degenerate primer with UPM because of high Tm difference. Could you suggest the solution for my problem?
@ Avnish......ypu can try touch down PCR. Hopefully you will get the results.
Hi Avneesh
Can you elaborate your RACE approach a little bit more....Which RACE your are planning to do : 3' or 5'...and what is size of product which you are expecting from the RACE reaction...
bye
I am doing both 5'&3'RACE. size of products are 900-1kb for 3' and 500-600 bp for 5' RACE.
@arun ... Sir ji...sab try kar liya...i cloned a lot of bands of size of interest but nothing positive......
Hi Avneesh
OK then what is the size of your gene (or cDNA)...for which you are planning RACE...if it is not known in your organism.....if something is known in other model organims..
Can you also give details of your approach for both the RACE reactions right from the begining...I may be able to give you some suggestions
bye
Hi Dear Avnish....send me the partial sequence and other details ...I will try to figure out the problem.
Hi Avneesh
I also agree with .....the quality of RNA should be very good for RACE and when you are trying for both 3' as well as 3' end RACE......the transcript of the gene of interest should be full length in the pool of RNAs....and second important thing will be quality of cDNA synthesis.....if the reverse transcriptase is not able to synthesize cDNA to its full length...getting the product in the RACE reaction will be difficult....
as you have also mentioned that you are able to amplify small lenght products but not full length products..so take care of these things...
bye
Hi Avneesh, sometimes is better to clone in E. coli bigger fragment taht includes the putative starting point you want to know. The kits for RACE are developed to used better in E. coli. Then check the qualitiy of RNA and quiantity.
I would presume RACE-PCR is always tricky. But i could clone and sequence the full-length sequence by 5'- and 3'-RACE-PCR in my first attempt. I started with designing RACE-PCR primers with my partial cDNA sequence of approx. 270 bp. I used the Clontech SMARTer RACE cDNA amplification kit. Both 5'- and 3'-RACE- ready cDNA was synthesized with an oligo-dT adaptor primer. Because of the terminal transferase activity of reverse transcriptase, the first strand cDNA possesses adaptor primer sequences. SNARTER IIA oligonucleotide was only added to 5'-RACE ready cDNA synthesis reaction. First PCR was carried out by the UPM and gene-specific reverse primer followed by nested PCR with nested gene specific reverse primer (for 5'-RACE). The nested PCR products were extracted and purified and subsequently cloned into the TA cloning vector and sequenced.
Continue with the nested PCR with GSP-2, from the first PCR product. Most often, it amplifies with a single band. The first PCR product may give you some unspecific bands, so please continue to the nested PCR.
Thanks to all for your valuable suggestions...there are something to discuss
I check RNA quality it was good.
I also cloned a lot of bands of desired size but always seq was from a different gene.
Markus Grohme ·dear sir i tried to amplify with only GSP and usethe adaptor primer (long & short) in two different reaction even with touchdown conditions. but nothing found. is there any other possible method to get full length seq of a gene.
Thanks to all for your valuable suggestions...there are something to discuss
I check RNA quality it was good.
I also cloned a lot of bands of desired size but always seq was from a different gene.
Markus Grohme ·dear sir i tried to amplify with only GSP and usethe adaptor primer (long & short) in two different reaction even with touchdown conditions. but nothing found. is there any other possible method to get full length seq of a gene.
I also had the same problem of not getting the sequence right after cloning. I presume you have to check for contamination maybe your insert or maybe the cell stock may be old. Sometimes satellite colonies can cause disturbances in trying to get the best clone. Good luck.
I am going to perform RACE-PCR. I don't know how to design GSP. Can any body help me?
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