Initially I had succesfully cloned 526bp, 425bp and a 256bp target cDNA into a pGEMT easy vector systems (Promega). Sequencing results were found to be positive on BLAST search. Then, in oder to proceed for full length cloning, I had tried RACE cloning using CLONTECH SMARTer RACE cDNA kit and Advantage 2 PCR kit (Clontech). 5' RACE PCR amplication resulted in a single band of 1100bp, while 3' RACE PCR of 1450bp when run on 1.5% agarose gel. Everything then on went fine, clones were screened by blue white screening method. Colony PCR was positve with both GSPs and T7 and SP6 universal primers. Plasmids were isolated and plasmid PCR with GSPs were positive showing single band at appropriate place. But sequencing results defied all results and didnt show any closer matching. I am stuck with this part (extension of 526bp target gene) very badly. I have tried both classical cloning with new primers and RACE (5' and 3') with both old and new primer sets but everytime met with failures. If anyone here can give valuable suggestions, I will be able to go a step further.
Did you sequence the 1100 and 1450 PCR product to determine if you had amplified the right mRNA? If the sequence of the PCR product is correct then you just need to work on the cloning part. If the sequence is not correct then you need to figure out why not and presumably design different primers.
Thanks Sir for replying. Yes I had sequenced both 5' and 3' RACE PCR clones of 1100bp and 1450bp respectively. But in case of 3' RACE PCR clone sequence showed no significant matches and in case of 5' RACE PCR clone, I got only ~550bp sequence that matched with target sequence which we already have confirmed initially (526bp successful target clone). The problem is I not able to extend that 526bp sequence. And yes we designed new primers also but I am facing the same problem.
I don't mean sequence the clones, I meant to sequence the actual PCR product before you clone it. That way you know if your source material for cloning is correct or not.
Thank you Michael. No, I haven't tried sequencing RACE PCR products. I should see that first. Thats a good point. Right now I am starting freshly again everything. I will get to you when I am done with it.
No Artur, its not human. The target gene belongs to fish. Yes I have considered sending PCR products for sequencing. Actually, I need to raise antibodies against the gene for the fish species I am working on. The NCBI database consists of the full length of the gene in other fish species, and my sequence results didn't match with those at all. But when I had initially performed classical cloning using three primer sets covering overlapping regions of the complete submitted gene sequence of other fish species, I had got 70-75% matches with the same. I had also used those primers for RT-PCR expression study, and have good results. But I am interested to start protein work to validate my study but as the antibodies for the target gene is not available commercially, I will have to generate the complete sequence, then raise antibodies and then could proceed with the protein work.
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